Anticoagulant peptide derivatives and pharmaceutical compositions containing the same as well as a process for preparation thereof

ABSTRACT

This invention relates to new peptide derivatives of the general formula (I) A-Xaa-Arg-H, wherein A represents a D- or L-isochroman-1-carbonyl, D- or L-isochroman-3-carbonyl group, furthermore an acyl group of the general formula: D- or DL-A&#39;--CH(OH)--CO, wherein A&#39; represents a phenyl, benzyl, 1-naphthyl, 1-naphthylmethyl, 2-naphthyl, 2-naphthylmethyl, 9-fluorenyl, benzhydryl, cyclohexyl, cyclohexylmethyl, 2-pyridyl, 3-pyridyl or 4-pyridyl group, and Xaa represents an L-prolyl or an L-pipecolinic acid residue, and Arg stands for an L-arginine residue, their acid addition salts formed with an organic or inorganic acid and pharmaceutical compositions containing the same. Furthermore the invention relates to a process for preparing them. The compounds of the invention have valuable therapeutic, particularly anticoagulant, properties.

This application is a continuation of application Ser. No. 08/302,724,filed on Dec. 5, 1994 now abandoned.

This invention relates to new peptide derivatives of the general formula(I),

    A-Xaa-Arg-H                                                (I)

wherein

A represents a D- or L-isochroman-1-carbonyl, D- orL-isochroman-3-carbonyl group, furthermore an acyl group of the generalformula

    D- or DL-A'--CH(OH)--CO,

wherein

A' represents a phenyl, benzyl, 1-naphthyl, 1-naphthylmethyl,2-naphthyl, 2-naphthylmethyl, 9-fluorenyl, benzhydryl, cyclohexyl,cyclohexylmethyl, 2-pyridyl, 3-pyridyl or 4-pyridyl group, and

Xaa represents an L-prolyl or an L-pipecolinic acid residue, and

Arg stands for an L-arginine residue,

their acid addition salts formed with an organic or inorganic acid andpharmaceutical compositions containing the same. The abbreviation of theL-arginine residue as Arg is in accordance with the prior art, e.g.Biochem. J. 126, 773 (1972); Biochemistry 14, 449 (1975)!.

The invention furthermore relates to a process for preparing the newpeptide derivatives of the general formula (I) and pharmaceuticalcompositions containing these compounds.

The compounds of the invention have valuable therapeutic, particularlyanticoagulant properties.

Preferred representatives of the compounds having the formula (I) arethose described in the Examples.

Particularly preferred representatives of the compounds according to theinvention are the following derivatives:

3-cyclohexyl-D-lactyl-L-prolyl-L-arginine aldehyde hemisulfate;

3,3-diphenyl-D-lactyl-L-prolyl-L-arginine aldehyde hemisulfate;

D-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehyde hemisulfate;

D-2-(1-naphthyl)-2-hydroxyacetyl-L-prolyl-L-arginine aldehydehemisulfate;

D-2-(2-naphthyl)-2-hydroxyacetyl-L-prolyl-L-arginine aldehydehemisulfate.

It is known that the aldehyde, derived from the tripeptideD-phenylalanyl-L-prolyl-L-arginine (Hungarian patent specification No.169,870 and U.S. Pat. No. 4,399,065), the trifluoromethyl-ketone Neisesand Tarnus: Thrombos Haemostas 65, 1290 (1991)!, and the boro-arginineKettner et al.: J. Biol. Chem. 265, 18289 (1990)! are potentanticoagulants exerting activity both in vitro and in vivo. However,these compounds are rather unstable and are converted in neutral aqueoussolution to inactive products Bajusz et al.: J. Med. Chem. 33, 1729(1990)!.

Further active tripeptide derivatives were prepared by acylating U.S.Pat. No. 4,478,475 and Kettner et al.: J. Biol. Chem. 265, 18289 (1990))or alkylating (U.S. Pat. No. 4,703,036) the D-phenylalanine moiety, orby exchanging it for analogue amino or imino acids such asN-methylphenylglycine or 1,2,3,4-tetrahydroisoquinoline-1-carboxylicacid Shuman et al.: Peptides, Proceedings of the Twelfth AmericanPeptide Symposium (Eds. J. A. Smith and J. E. Rivier), ESCOM, Leyden,pp. 801-802, 1992!. It is a characteristic feature of highly activeanticoagulant peptides that they are tripeptide derivatives, and theN-terminal residue, linked to the central L-proline moiety, is an aminoor imino acid residue. These tripeptides correspond to the generalformula (I) only if A stands for a D-phenylalanine residue, and acylatedor alkylated derivative an amino acid or imino acid analogue thereof.

It is the objective of the present invention to provide novel peptidederivatives of improved stability and bioavailability compared to knowncompounds.

It was unexpectedly found that the tripeptide structure is not aprerequisite of anticoagulant activity, a carboxylic acid groupcontaining an oxygen atom in the alpha-position A in general formula(I)! can be advantageously substituted for the N-terminal amino acid orimino acid moiety of the known compounds.

Furthermore it has also been found that the compounds of the generalformula (I) according to the invention, wherein A, A', Xaa and Arg havethe same meaning as above, and their acid-addition salts are stable inaqueous solution, exert strong anticoagulant activity both in vitro andin vivo, and have favorable bioavailability.

According to the invention the compounds of general formula (I) andtheir acid-addition salts formed with an organic or inorganic acid areprepared by a process which comprises condensing an acid, containing anacyl radical A, wherein A has the above meaning, and L-proline orL-pipecolinic acid, converting the thus-obtained acyl-L-proline oracyl-L-pipecolinic acid by a method known in the art by acylating anarginine lactam protected at the guanidino group with the saidacyl-L-proline or acyl-L-pipecolinic acid, reducing the protectedacyl-arginine lactam to the protected acyl-arginine aldehyde andremoving the protecting groups, and, if desired, finally converting theresulting compound of the general formula (I) with an inorganic ororganic acid to an acid-addition salt.

Compounds of the general formula (I), wherein A, A', Xaa and Arg havethe same meaning as in the introduction, are prepared from the newacyl-L-proline or acyl-L-pipecolinic acid synthesized by the first stepof the process of the invention, according to the method as described inthe U.S. Pat. No. 4,703,036 disclosing compounds of similar structure.

According to a preferred embodiment of the process according to theinvention DL-isochroman-1-carboxylic acid is condensed with L-prolinet-butyl ester, the ester group is removed by acidolysis from theDL-acyl-L-proline t-butyl ester formed, then the product is submitted tosilica gel column chromatography to separate D- andL-isochroman-1-carbonyl-L-prolin, subsequentlyD-isochroman-1-carbonyl-L-proline is condensed with N^(G)-benzyloxycarbonyl-L-arginine lactam, the thus-obtainedD-iso-chroman-1-carbonyl-L-prolyl-N^(G) -benzyloxycarbonyl-L-argininelactam is reduced with lithium aluminium hydride to the respectivealdehyde, the N^(G) protecting group is removed by hydrogenolysis inaqueous ethanol solution containing sulfuric acid in the presence of aPd--C catalyst, the catalyst is filtered off and the product isseparated by freeze-drying.

According to an other preferred embodiment of the present inventionD-mandelic acid methyl ester is reacted with dihydropyran, theO-tetrahydropyranyl-D-2-phenyl-2--hydroxyacetic acid methyl ester formedis saponified, then condensed with L-proline methyl ester, the resultingO-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-proline methyl ester issaponified, thereafter condensed with N^(G)-benzyloxycarbonyl-L-arginine lactam and the obtainedO-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam is reduced with lithium aluminiumhydride to the respective aldehyde, the protecting groups are removed bydissolving the product in aqueous ethanol containing sulfuric acid andby submitting this solution to catalytic hydrogenation in the presenceof Pd--C, filtering off the catalyst and isolating the product byfreeze-drying.

According to a further preferred embodiment of the present inventionDL-2-(2-naphthyl)-2-hydroxyacetic acid methyl ester is converted withisobutene to O-t-butyl-DL-2-(2-naphthyl)-2-hydroxyacetic acid methylester, then saponified, the O-t-butyl-DL-2-(2-naphthyl)-2-hydroxyaceticacid obtained is condensed with L-proline methyl ester, the resultingdiastereomer pair ofO-t-butyl-D-2-(2-naphthyl)-2-hydroxyacetyl-L-proline methyl ester andO-t-butyl-L-2-(2-naphthyl)-2-hydroxyacetyl-L-proline methyl ester issaponified, separated on an anion-exchange resin (acetate cycle), theO-t-butyl-D-2-(2-naphthyl)-2-hydroxyacetyl-L-proline obtained as firstproduct when eluting the column is condensed with N^(G)-benzyloxycarbonyl-L-arginine lactam, the obtainedO-t-butyl-D-2-(2-naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam, after acidic deblocking of thet-butyl group, is reduced with lithium aluminium hydride to therespective aldehyde, the N^(G) -protecting group is removed in aqueousethanol solution containing sulfuric acid by catalytic hydrogenation inthe presence of Pd--C, the catalyst is filtered off and the product isisolated by freeze-drying.

According to a further preferred embodiment of the present inventioncompounds of the general formula (I), wherein Xaa stands for anL-pipecolic acid residue, are prepared by condensing acyl-L-pipecolicacid and N^(G) -benzyloxycarbonyl-L-arginine lactam in pyridine in thepresence of phosphorus oxychloride, the acyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam obtained is reduced to thecorresponding aldehyde, then after deblocking the last protecting groupthe product is isolated by freeze-drying.

In the acid-addition salts of the peptide derivatives of general formula(I) the activity resides in the base and the acid is of less importancealthough for therapeutic purposes it is preferable to usepharmacologically and pharmaceutically acceptable acid-addition salts.Examples of such suitable acids include (a) mineral acids: hydrochloric,hydrobromic, phosphoric, metaphosphoric and sulphuric acids, (b) organicacids: tartaric, acetic, citric, malic, lactic, fumaric, benzoic,glycolic, gluconic, succinic, pamoic and aryl sulphonic acids, forexample p-toluenesulphonic acid.

The antithrombin activity of the compounds of general formula (I) wasdetermined by the usual method Bagdy et al.: Thromb Haemostas 67,325-330 (1992)! by measuring whole blood clotting time (WBCT), thrombintime (TT) and activated partial thromboplastin time (APTT). The in vitroactivity of some compounds of the invention assayed on native humanblood (non-anticoagulated clotting) (WBCT) and on citrated blood plasma(TT and APTT) is presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        In vitro antithrombin effect of new                                           peptide derivatives of general formula (I)                                    A-Xaa-Arg-H.sup.1                                                             Example                    Clotting parameters.sup.2                          No.      A          Xaa    WBCT    TT   APTT                                  ______________________________________                                        1        D-Ica (1)  Pro    1.55    0.132                                                                              0.60                                  6        D-Man      Pro    0.45    0.040                                                                              0.15                                  7        D-Hpl      Pro    0.32    0.072                                                                              0.20                                  8        D-Hma      Pro    0.72    0.100                                                                              0.35                                  12       D-Dpl      Pro    0.52    0.077                                                                              0.12                                  13       D-Nga (1)  Pro    1.13    0.100                                                                              1.00                                  14       D-Nga (2)  Pro    1.04    0.150                                                                              0.52                                  ______________________________________                                         .sup.1 Abbreviations: Dpl = 3,3diphenyl-lactyl, Hma =                         cyclohexyl2-hydroxyacetyl, Hpl = 3cyclohexyl-lactyl, Ica (1) =                isochroman1-carbonyl, Man = 2phenyl-2-hydroxyacetyl, Nga (1) =                2(1-naphthyl)-2-hydroxyacetyl, Nga (2) = 2(2-naphthyl)-2-hydroxyacetyl,       Pro = Lprolyl.                                                                .sup.2 WBCT = whole blood clotting time, TT = thrombin time, APTT =           activated partial thromboplastin time parameter. The figures represent        concentrations of the compounds required to double the clotting time of       native whole human blood or of citrated blood plasma.                    

The in vivo antithrombin effect of the compounds of general formula (I)was treated in rabbits by a known method Bagdy et al: Thromb Haemost 67,357-365 (1992)!. The compounds were applied in a dose of 0.5-3.0 mg/kgs.c. and 5-15 mg/kg p.o. The effect was already detectable 15-30 minutesafter drug administration, peak effect was attained after 90-120 minutesbut the therapeutic effect was dose dependently maintained during 3 tolonger than 5 hours.

The in vivo antithrombin effect of3-cyclohexyl-D-lactyl-L-prolyl-L-arginine aldehyde hemisulfate (Example7.) is presented in detail in Tables 2 and 3. The compound wasadministered in doses of 10 and 15 mg/kg, resp., p.o. and the bloodsamples taken from the ear-vein of the animals were assayed in every 30minutes. The whole blood clotting time (WBCT) and the inhibition ofwhole blood platelet aggregation induced by thrombin (IWBA) weredetermined. Furthermore the citrated plasma thrombin time (TT) of theblood sample and the activated partial thromboplastin time (APTT) weremeasured, too. The WBCT and APTT values are presented in Table 2 and theTT and IWBA values in Table 3.

                  TABLE 2                                                         ______________________________________                                        Antithrombin effect of 10 and 15 mg/kg p.o. doses of                          3-cyclohexyl-D-lactyl-L-prolyl-L-arginine aldehyde                            hemisulfate in rabbits. WBCT and APTT values                                  Time   10 mg/kg         15 mg/kg                                              (min.) WBCT (min.)                                                                              APTT (sec)                                                                              WBCT (min.)                                                                            APTT (sec)                               ______________________________________                                        0      13.2 ± 1.1                                                                            27.7 ± 1.2                                                                           14.6 ± 0.4                                                                          32.6 ± 4.6                            30     17.7 ± 1.6                                                                            58.9 ± 9.0                                                                           24.2 ± 4.7                                                                          115.0 ± 36.6                          60     23.6 ± 1.9                                                                             77.6 ± 14.6                                                                         29.4 ± 4.5                                                                          150.4 ± 30.9                          90     21.2 ± 2.9                                                                            65.1 ± 8.4                                                                           33.0 ± 5.9                                                                          170.3 ± 18.5                          120    18.8 ± 1.0                                                                             55.1 ± 11.4                                                                         34.4 ± 4.4                                                                          145.6 ± 22.7                          150    19.2 ± 1.1                                                                            52.1 ± 6.6                                                                           27.2 ± 3.6                                                                          149.2 ± 22.0                          180    18.4 ± 1.5                                                                            53.8 ± 8.5                                                                           29.5 ± 2.7                                                                          169.0 ± 23.0                          210    17.2 ± 2.3                                                                            48.6 ± 3.8                                                                           34.0 ± 5.9                                                                          137.7 ± 25.8                          240    15.8 ± 1.7                                                                            47.7 ± 7.4                                                                           33.7 ± 6.5                                                                          127.4 ± 25.7                          270    16.2 ± 2.1                                                                            44.4 ± 3.7                                                                           26.0 ± 1.2                                                                          133.4 ± 24.7                          300    13.5 ± 0.9                                                                            43.7 ± 5.0                                                                           23.6 ± 3.3                                                                           92.8 ± 18.4                          ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Antithrombin effect of 10 and 15 mg/kg p.o. doses of                          3-cyclohexyl-D-lactyl-L-prolyl-L-arginine aldehyde                            hemisulfate in rabbits. TT and IWBA values                                    Time  10 mg/kg         15 mg/kg                                               (min.)                                                                              TT (sec)   IWBA (%)  TT (sec)  IWBA (%)                                 ______________________________________                                        0     18.4 ± 2.1        18.9 ± 2.1                                      30    317.1 ± 123.0                                                                         89.4 ± 7.6                                                                            371.2 ± 142.3                                                                       81.4 ±                                                                     11.7                                     60    416.0 ± 107.2                                                                         95.8 ± 6.9                                                                           528.3 ± 72.9                                                                         97.4 ±                                                                     2.6                                      90    444.7 ± 108.6                                                                         81.6 ± 8.2                                                                           578.8 ± 21.5                                                                         100.0 ± 0                             120   264.6 ± 101.3                                                                         94.0 ± 3.2                                                                           >600.0 ± 0                                                                           100.0 ± 0                             150   201.1 ± 109.9                                                                         71.4 ± 13.8                                                                          549.6 ± 34.0                                                                         100.0 ± 0                             180   182.7 ± 113.6                                                                         72.4 ± 19.2                                                                           380.7 ± 116.0                                                                       100.0 ± 0                             210   123.0 ± 65.3                                                                          54.4 ± 21.0                                                                           342.1 ± 121.2                                                                       93.7 ±                                                                     4.9                                      240   141.5 ± 81.3                                                                          50.6 ± 17.7                                                                           340.9 ± 137.0                                                                       82.6 ±                                                                     17.7                                     270   65.4 ± 25.7                                                                           35.2 ± 17.3                                                                           376.4 ± 153.0                                                                       81.0 ±                                                                     16.9                                     300   41.8 ± 11.4                                                                           48.2 ± 16.9                                                                           261.0 ± 140.8                                                                       69.6 ±                                                                     20.4                                     ______________________________________                                    

The compounds of the general formula (I) inhibit clot formation withoutinterfering with the natural lysis of blood clots in the body, e.g.fibrinolysis. This was confirmed by the fact that the compounds ofExamples 6, 7 and 8 had only negligible inhibitory effect onfibrinolysis induced by tissue plasminogen activator measured in athromboelastogram H. Hartert. Thromboelastography. In: Bang N. U., F. K.Beller, E. Deutsch, E. F. Mamman eds. Thrombosis and Bleeding Disorders.Theory and Methods. Academic Press, London, 1971: 70-76!.

The invention in one of its aspects provides a method for inhibiting theformation of blood clots in man and animals which comprisesadministering to said man or animal an effective clot inhibitingnon-toxic dose of a compound represented by the formula (I). Theanticoagulant compound is administered orally, parenterally, e.g. byintravenous infusion (i.v.), intramuscular injection (i.m.) orsubcutaneously (s.c.). Preferably administration is carried out bysubcutaneous injection or orally.

An effective clot inhibiting dose is 0.3 to 3.0 mg/kg, preferably 0.5 to1.0 mg/kg s.c. and 3 to 20 mg/kg, preferably 5 to 15 mg/kg p.o. The doseregime may vary according to the age and state of the patient, e.g. forprophylactic use a single daily dose may be administered or multipledoses such as 3 or 5 times daily may be appropriate.

The method of this invention is practiced in conjunction with a clotlyzing agent, e.g. tissue plasminogen activator (tPA), modified tPA,streptokinase or urokinase. In cases when clot formation has occurredand an artery or vein is blocked partially or totally, a clot lyzingagent is usually employed. A compound of the invention can beadministered along with the lyzing agent or subsequent to its use toprevent the reoccurrence of clot formation.

In carrying out the method the use of a preferred compound of theinvention is desirable. For example use is made of a preferred compoundsuch as described hereinabove. Preferred peptide derivatives are3-cyclohexyl-D-lactyl-L-prolyl-L-arginine aldehyde hemisulfate,3,3-diphenyl-D-lactyl-L-prolyl-L-arginine aldehyde hemisulfate andD-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehyde hemisulfate.

The invention also provides pharmaceutical formulations for use in theabove-described therapeutic method. Pharmaceutical formulations of theinvention comprise an effective clot inhibitory amount of a compound ofthe formula (I) or of a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier. For oral administration theantithrombotic compound is formulated in gelatine capsules or tabletswhich may contain excipients such as binders, lubricants, disintegrationagents and the like. For parenteral administration the antithromboticcompound is formulated in a pharmaceutically acceptable diluent, e.g.physiological saline (0.9%), 5% dextrose, Ringer's solution and thelike.

The following examples are illustrating but not limiting the scope ofthe invention.

The R_(f) values recorded in the examples were determined by thin-layerchromatography, using silica gel as adsorbent (DC-Alufolien Kieselgel 60F₂₅₄, Merck, Darmstadt) in the following developing solvents:

1. Ethyl acetate-pyridine-acetic acid-water (480:20:6:11)

2. Ethyl acetate-pyridine-acetic acid-water (240:20:6:11)

3. Ethyl acetate-pyridine-acetic acid-water (120:20:6:11)

4. Ethyl acetate-pyridine-acetic acid-water (60:20:6:11)

5. Ethyl acetate-pyridine-acetic acid-water (45:20:6:11)

6. Ethyl acetate-pyridine-acetic acid-water (30:20:6:11)

7. Ethyl acetate-pyridine-formic acid-water (480:20:6:5.5)

8. Ethyl acetate-pyridine-formic acid-water (240:20:6:5.5)

9. Ethyl acetate-pyridine-acetic acid-water (960:20:6:11)

10. Ethyl acetate-diisopropyl ether (7:3)

11. Ethyl acetate-n-hexane (1:1)

12. Chloroform-acetone (98:2).

The capacity factors specified in the examples were determined with theapparatus "Pharmacia LKB analytical HPLC System Two" as follows.

Column I

"VYDAC C-18 reversed phase: 10 μm, 300 Å, 4×250 mm".

Buffer A: 0.1% trifluoroacetic acid in water

Buffer B: 0.1% trifluoroacetic acid in acetonitrile

Column II

Mono Q HR 5/5 (Pharmacia).

Buffer A: Methanol-water (2:1)

Buffer B: Methanol-0.2M NaCl (2:1)

Gradients applied (flow rate):

1. 0-5 min.: 0-15% B; 5-30 min.: 15% B (1 ml/min.);

2. 0-5 min.: 0-15% B; 5-30 min.: 15% B (1.2 ml/min.);

3. 0-30 min.: 0-60% B; (1 ml/min.);

4. 0-5 min.: 0-6% B; 5-30 min.: 6% B; 30-35 min.: 6-18% B (1.2 ml/min.);

5. 0-5 min.: 0% B, 5-30 min.: 2% B (0.8 ml/min.).

The peptide content of the eluate was detected in UV light at 214 nm.Sample concentration: 1 mg/ml buffer A, injection volume: 25 μl.

Conditions of the HPLC analysis, the column and buffers (I or II)applied as well as the data of the gradients (1-5) are specified afterthe abbreviation in brackets, for instance HPLC(II/5).

The acyl-arginine aldehydes are present in equilibrium structures, i.e.in aldehyde, aldehyde hydrate and two aminocyclol forms. During HPLCanalysis the aldehyde hydrate and one or both aminocyclol forms appearas separate peaks, consequently the products described in the examplescan be specified by two or three k' values.3-Cyclohexyl-D-lactyl-L-pipecolyl-L-arginine aldehyde hemisulfate ofExample 9 represents an exception where six k' values corresponding tothe six peaks are specified which are presumably due to the knowncis-trans isomerization of the acyl-pipecolines.

EXAMPLE 1 D-Isochroman-1-carbonyl-L-prolyl-L-arginine aldehydehemisulfate

Step 1: D-Isochroman-1-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

2.73 g (7 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 331729 (1990)! is suspended in 10 ml of chloroform, then 10 ml of ethylacetate, saturated with HCl gas (0.11-0.15 g/ml), is added at constantstirring and ice cooling. After 2 hours the reaction mixture is dilutedwith 10-15 ml of diethyl ether, the crystal mass formed is filtered,washed with 5 ml of acetone and 5 ml of diethyl ether, then dried overpotassium hydroxide at reduced pressure overnight. The N^(G)-benzyloxycarbonyl-L-arginine lactam hydrochloride obtained is dissolvedin 10 ml of dimethyl formamide, cooled to -20° C. and added to the mixedanhydride below.

2.25 g (6 mmole) of D-isochroman-1-carbonyl-L-proline cyclohexylammoniumsalt (Example 1, Step D) is dissolved in 10 ml of ethyl acetate and 10ml of 1M potassium hydrogen sulfate. The ethyl acetate layer is driedover sodium sulfate, then evaporated at 20-25 millibar from a water bathof about 40° C., finally further 5 ml of benzene is distilled off theresidue which is dissolved in 6 ml of dimethyl formamide. The obtainedsolution is cooled to -20° C., then 0.66 ml (6 mmole) ofN-methylmorpholine and 0.79 ml of isobutyl chloroformate are added atstirring. Stirring is continued for 10 minutes, then the abovesuspension of N^(G) -benzyloxycarbonyl-L-arginine lactam in dimethylformamide and finally 2.1 ml (15 mmole) of triethylamine are added. Thereaction mixture is stirred for 2 hours first at cooling, later it isleft to warm up to room temperature. Thereafter the salt is filteredoff, the filtrate is diluted with 50 ml of benzene, washed with 4×20 mlof water, 3×10 ml of 0.1M hydrochloric acid and again with 3×20 ml ofwater, dried over sodium sulfate and evaporated at 20-25 millibar from awater bath of about 40° C. The residue is dissolved in 1.5 ml of amixture of ethyl acetate:pyridine:formic acid:water (240:20:6:5.5),transferred to a column prepared from 100 g of silica gel with ethylacetate and eluted with a mixture of ethyl acetate:pyridine:formicacid:water (480:20:6:5.5). The fractions containing solely the pureproduct are pooled, shaken with 1/3 volume of 1M potassium hydrogencarbonate, the organic layer is washed to neutrality with water, driedover sodium sulfate and evaporated at 20-25 millibar from a water bathof about 40° C. The residue is dissolved in benzene, then repeatedlyevaporated and treated with n-hexane.

Yield 2.3 g (70%), R_(f) (7)=0.42-0.46

α!_(D) ²⁰ =-28.5° (c=1, tetrahydrofuran)

Analysis for C₂₉ H₃₃ N₅ O₆ (547.59) Calculated: C %=63.60; H %=6.07; N%=12.79; Found: C %=63.5; H %=6.1; N %=12.5.

Step 2: D-Isochroman-1-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

1.64 g (3 mmole) of D-isochroman-1-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 1, Step 1) is dissolved in8 ml of tetrahydrofuran then 2.25 mmole of lithium aluminium hydride,dissolved in tetrahydrofuran, is added at a temperature not higher than-20° C. and at constant stirring. The progress of reduction is monitoredby thin-layer chromatography using a developing solvent system of ethylacetate:pyridine:acetic acid:water (240:20:6:11) and, if required, afurther portion of lithium aluminium hydride is added. Then the reactionmixture is poured under cooling and stirring into 20 ml of cold 1Mpotassium hydrogen sulfate. The solution is diluted with 10 ml of water,then extracted with 2×10 ml of n-hexane and 3×10 ml of methylenechloride. The methylene chloride solutions are pooled, washed with 3×5ml of water, 5% aqueous sodium hydrogen carbonate and again with water,dried over sodium sulfate and evaporated at 20-25 millibar from a waterbath at about 40° C. The evaporation residue is treated with n-hexane.

Yield 1.15 g (70%), R_(f) (3)=0.50-0.52.

Step 3: D-Isochroman-1-carbonyl-L-prolyl-L-arginine aldehyde hemisulfate

0.82 g (1.5 mmole) of D-isochroman-1-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 1, Step 2) is dissolvedin 10 ml of 60% aqueous ethanol containing 0.75 mmole of sulfuric acid,then hydrogenated in the presence of Pd--C catalyst. The progress of thereaction is monitored by thin-layer chromatography in a developingsolvent system of ethyl acetate:pyridine:acetic acid:water (45:20:6:11).The reaction is completed in about 30 minutes, then the catalyst isfiltered off and washed with 60% aqueous ethanol and water. The filtrateis evaporated to about half of its volume at reduced pressure, then itis diluted with water to 10 ml. The solution is first extracted withmethylene chloride, then it is freeze-dried. Yield: 0.5 g (70%).

R_(f) (5)=0.48-0.52; HPLC(I/1): k'=3.95, 4.76, 5.95.

α!_(D) ²⁰ =-70.86° (c=1, 0.1M hydrochloric acid)

FAB mass spectrum (416 M+H!⁺) confirms the assumed structure.

The starting materials can be prepared as follows:

D-Isochroman-1-carbonyl-L-proline cyclohexylammonium salt

Step A: DL-Isochroman-1-carboxylic acid

12 ml (0.1 mole) of phenethyl alcohol and 10.12 g (0.11 mole) ofglyoxylic acid hydrate are dissolved in 50 ml of trifluoroacetic acid.The solution is refluxed during 24 hours, then evaporated at 20-25millibar from a water bath at max. 40° C. 100 ml of water andconcentrated ammonium hydroxide in a volume sufficient to adjust the pHof the solution over 7 are added to the residue. The solution isextracted with 3×30 ml of diethyl ether, its pH is adjusted to about 3with saturated potassium hydrogen sulfate, then the solution isextracted with 3×30 ml of ethyl acetate. The ethyl acetate solutions arepooled, washed to neutrality with water (3-4×20 ml), dried over sodiumsulfate and evaporated at 20-25 millibar from a water bath at about 40°C. The residue is treated with n-hexane, filtered, washed with n-hexaneand air-dried.

Yield 10.7-11.6 g (60-65%)

R_(f) (3)=0.65-0.75; m.p.: 92°-93° C.

Analysis for C₁₀ H₁₀ O₃ (178.18) Calculated: C %=67.40; H %=5.66; Found:C %=67.45; H %=5.65.

Step B: DL-Isochroman-1-carbonyl-L-proline t-butyl ester

4.45 g (25 mmole) of DL-isochroman-1-carboxylic acid (Example 1, Step A)and 2.75 ml (25 mmole) of N-methylmorpholine are dissolved in 20 ml ofdimethyl formamide. The solution is cooled to -15° C., then at thistemperature and at stirring, first 3.3 ml of isobutyl chloroformate,then, after 10 minutes, the solution or suspension of 5.2 g (25 mmole)of L-proline t-butyl ester hydrochloride and 3.5 ml of triethylamine in25 ml of dimethyl formamide, cooled to -15° C., are added. Stirring iscontinued for 2 hours, then the reaction mixture is filtered andevaporated at 15-20 millibar from a water bath at about 40° C. Theresidue is dissolved in about 30 ml of ethyl acetate and the solutionare extracted with 3×20 ml of 0.25M sulfuric acid, water, 5% sodiumhydrogen carbonate, again with water, dried over sodium sulfate andevaporated at 20-25 millibar from a water bath of about 40° C. Theresidue-DL-isochroman-1-carbonyl-L-proline t-butyl ester--is an oilyproduct.

R_(f) (7)=0.72 and 0.75!.

Step C: DL-Isochroman-1-carbonyl-L-proline

The oily product prepared according to Example 1, Step B is dissolved in25 ml of trifluoroacetic acid and left to stand at room temperature for2 hours. Thereafter the solution is evaporated at 20-25 millibar from awater bath of about 40° C., the residue is dissolved in 30 ml of waterand repeatedly evaporated. The residue obtained is dissolved in 50 ml ofethyl acetate and extracted with 3×30 ml of 5% sodium hydrogencarbonate. The sodium hydrogen carbonate solutions are pooled, its pH isadjusted to 3 with 1M potassium hydrogen sulfate and the solution isextracted with 3×30 ml of ethyl acetate. The ethyl acetate layers arepooled, dried over sodium sulfate and evaporated. The oily residue isthe diastereomer pair comprising D- andL-isochroman-1-carbonyl-L-proline.

Yield 25 mmole, R_(f) (8)=0.40-0.42 and 0.51-0.53, resp.

Step D: D- and L-isochroman-1-carbonyl-L-proline cyclohexylammonium salt

The diastereomer pair obtained in Example 1, Step C is separated bysilica gel column chromatography, applying 350 g of adsorbent and aneluent of ethyl acetate:pyridine:formic acid:water (480:20:6:5.5). Thefractions containing solely compound F top spot R_(f) (8)=0.51-0.53! andsolely compound A bottom spot R_(f) (8)=0.40-0.42! are pooled andprocessed by an identical method as follows.

The solution is evaporated in the usual manner (20-25 millibar, waterbath temperature about 40° C.) to dryness and the residue is dissolvedin 30 ml of methylene chloride. The thus-obtained solution is washedwith 15% aqueous sodium chloride solution, dried and evaporated. Theresidual oil is dissolved in 20 ml of diethyl ether, thencyclohexylamine is added in an amount to render the solution mildlybasic. The precipitated crystals of the cyclohexylammonium salt arefiltered, washed with diethyl ether and dried under reduced pressureover concentrated sulfuric acid.

The product isolated from solution F is, according to its NMR spectrum,D-isochroman-1-carbonyl-L-proline cyclohexylammonium salt.

Yield 2.75 g (58.75%).

R_(f) (8)=0.54-0.59; m.p.: 122°-125° C.;

α!_(D) ²⁰ =-41.5° (c=1, methanol).

Analysis for C₂₁ H₃₀ N₂ O₄ (374.47) Calculated: C %=67.35; H %=8.07; N%=7.48; Found: C %=67.3; H %=8.15; N %=7.24.

The product isolated from solution A is, according to its NMR spectrum,L-isochroman-1-carbonyl-L-proline cyclohexylammonium salt.

Yield 3.1 g (66.3%).

R_(f) (8)=0.36-0.41; m.p.: 154°-157° C.;

α!_(D) ²⁰ =-51.15° (c=1, methanol).

Analysis for c₂₁ H₃₀ N₂ O₄ (374.47) Calculated: C %=67.35; H %=8.07; N%=7.48; Found: C %=66.39; H %=8.45; N %=7.435.

EXAMPLE 2 L-Isochroman-1-carbonyl-L-prolyl-L-arginine aldehydehemisulfate

Step 1: L-Isochroman-1-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

Applying 2.73 g (7 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 33,1729 (1990)! and 2.25 g (6 mmole) of L-isochroman-1-carbonyl-L-prolinecyclohexylammonium salt (Example 1, Step D) as starting compounds, andusing the process described in Example 1, Step 1 in every respect,utilizing identical amounts of solvents and reagents, 2.3 g (70%) of theaimed product is obtained.

R_(f) (7)=0.42-0.46.

α!_(D) ²⁰ =-70.3° (c=1, tetrahydrofuran)

Analysis for C₂₉ H₃₃ N₅ O₆ (547.59) Calculated: C %=63.60; H %=6.07; N%=12.79; Found: C %=63.3; H %=6.2; N %=12.4.

Step 2: L-Isochroman-1-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

1.64 g (3 mmole) of L-isochroman-1-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 2, Step 1) is transformedby the process described in Example 1, Step 2, using identical amountsof solvents and reagents.

Yield 1.15 g (70%)

R_(f) (3)=0.48-0.50.

Step 3: L-Isochroman-1-carbonyl-L-prolyl-L-arginine aldehyde hemisulfate

0.82 g (1.5 mmole) of L-isochroman-1-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 2, Step 2) istransformed by the process described in Example 1, Step 3, usingidentical amounts of solvents and reagents.

Yield 0.5 g (70%)

R_(f) (5)=0.48-0.52; HPLC(I/1): k'=3.90, 4.62, 5.29.

α!_(D) ²⁰ =-55.2° (c=1, 0.1M hydrochloric acid).

FAB mass spectrum (416 M+H!⁺) confirms the assumed structure.

EXAMPLE 3 D-Isochroman-3-carbonyl-L-prolyl-L-arginine aldehydehemisulfate

Step 1: D-Isochroman-3-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

Applying 2.73 g (7 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 33,1729 (1990)! and 2.25 g (6 mmole) of D-isochroman-3-carbonyl-L-prolinecyclohexylammonium salt (Example 3, Step C) as starting compounds, andusing the process described in Example 1, Step 1 in everyrespect--utilizing identical amounts of solvents and reagents--yields2.3 g (70%) of the product.

R_(f) (9)=0.45-0.55.

α!_(D) ²⁰ =-50° (c=1, tetrahydrofuran)

Analysis for C₂₉ H₃₃ N₅ O₆ (547.59) Calculated: C %=63.60; H %=6.07; N%=12.79; Found: C %=63.8; H %=6.2; N %=12.5.

Step 2: D-Isochroman-3-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

2.2 g (4 mmole) of D-isochroman-3-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 3, Step 1) is transformedby the process described in Example 1, Step 2, using identical amountsof solvents and reagents.

Yield 1.55 g (70%)

R_(f) (2)=0.15-0.25.

α!_(D) ²⁰ =-28.6° (c=1, tetrahydrofuran)

Step 3: D-Isochroman-3-carbonyl-L-prolyl-L-arginine aldehyde hemisulfate

1.1 g (2 mmole) of D-isochroman-3-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 3, Step 2) istransformed by the process described in Example 1, Step 3, usingidentical amounts of solvents and reagents.

Yield 0.67 g (70%)

R_(f) (4)=0.20-0.30; HPLC(I/1): k'=4.55, 5.65, 6.95.

FAB mass spectrum (416 M+H!⁺) confirms the assumed structure.

The starting materials can be prepared as follows:

D-Isochroman-3-carbonyl-L-proline cyclohexylammonium salt

Step A: D-Isochromane-3-carboxylic acid

To 3 g of paraformaldehyde dissolved in 50 ml of trifluoroacetic acid16.6 g (0.1 mole) of 3-phenyl-D-lactic acid M. Winitz et al.: J. Am.Chem. Soc. 82 2423 (1956)! is added. The solution is refluxed for 48hours, then it is evaporated at 20-25 millibar from a water bath ofabout 40° C. Further 50 ml of water is distilled off, thereafter thecrystalline product formed is recrystallized from water. The crystalsare filtered, washed with water and dried at reduced pressure overphosphorus pentoxide.

Yield 14.2-16.0 g (80-90%)

R_(f) (1)=0.40-0.50; m.p.: 86°-88° C.

α!_(D) ²⁰ =+149° (c=1, methanol)

Analysis for C₁₀ H₁₀ O₃ (178.18) Calculated: C %=67.4; H %=5.66; Found:C %=67.4; H %=5.65;

Step B: D-Isochroman-3-carbonyl-L-proline t-butyl ester

4.45 g (25 mmole) of D-isochroman-3-carboxylic acid (Example 3, Step A)and 5.2 g (25 mmole) of L-proline t-butyl ester hydrochloride arecondensed according to the process described in Example 1, Step B,applying identical amounts of solvents and reagents. The oily product isD-isochroman-3-carbonyl-L-proline t-butyl ester.

Yield 25 mmole.

R_(f) (1)=0.80-0.90.

Step C: D-Isochroman-3-carbonyl-L-proline cyclohexylammonium salt

The oily product of Example 3, Step B is dissolved in 25 ml oftrifluoroacetic acid and left to stand at room temperature for 2 hours.The trifluoroacetic acid solution is evaporated at 20-25 millibar from awater bath at about 40° C., the residue is dissolved in 30 ml of waterand repeatedly evaporated. 50 ml of water and solid sodium hydrogencarbonate in an amount to adjust the pH over 7 are added to the oilyresidue. The solution is extracted with 3×10 ml of ethyl acetate, thenthe solution is acidified to pH 3 with 1M potassium hydrogen sulfate andextracted with 3×30 ml of ethyl acetate. After drying over sodiumsulfate the ethyl acetate extracts are evaporated. The oily evaporationresidue is dissolved in 20 ml of diethyl ether, then cyclohexylamine isadded in an amount to render the solution slightly alkaline. Theprecipitated crystal mass of the cyclohexylammonium salt is filtered,washed with diethyl ether and dried at reduced pressure overconcentrated sulfuric acid.

Yield 6.5 g (70%)

R_(f) (1)=0.30-0.40; m.p.: 158°-160° C.

α!_(D) ²⁰ =-3.5° (c=1, methanol)

Analysis for C₂₁ H₃₀ N₂ O₄ (374.47) Calculated: C %=67.35; H %=8.07; N%=7.48; Found: C %=67.4; H %=8.4; N %=7.3.

EXAMPLE 4 L-Isochroman-3-carbonyl-L-prolyl-L-arginine aldehydehemisulfate

Step 1: L-Isochroman-3-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

Applying 2.73 g (7 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 33,1729 (1990)! and 2.25 g (6 mmole) of L-isochroman-3-carbonyl-L-prolinecyclohexylammonium salt (Example 4, Step C) as starting compounds andusing the process described in Example 1, Step 1 in everyrespect--utilizing identical amounts of solvents and reagents--yields2.3 g (70%) of the aimed product.

R_(f) (9)=0.50-0.60.

Step 2: L-Isochroman-3-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

2.2 g (4 mmole) of L-isochroman-3-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 4, Step 1) is transformedby the process described in Example 1, Step 2, using proportionalamounts of solvents and reagents.

Yield 1.55 g (70%)

R_(f) (2)=0.20-0.30.

Step 3: L-Isochroman-3-carbonyl-L-prolyl-L-arginine aldehyde hemisulfate

1.1 g (2 mmole) of L-isochroman-3-carbonyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 4, Step 2) istransformed by the process described in Example 1, Step 3, usingproportional amounts of solvents and reagents.

Yield 0.67 g (70%)

R_(f) (4)=0.25-0.35; HPLC(I/1): k'=4.65, 5.40, 6.65.

FAB mass spectrum (416 M+H!⁺) confirms the assumed structure.

The starting materials can be prepared as follows:

L-Isochroman-3-carbonyl-L-proline cyclohexylammonium salt

Step A: L-Isochroman-3-carboxylic acid

1.5 g of paraformaldehyde and 8.3 g (50 mmole) of 3-phenyl-L-lactic acidM. Winitz et al.: J. Am. Chem. Soc. 82, 2423 (1956)! are condensedaccording to the process described in Example 3, Step A, usingproportional amounts of solvents and reagents.

Yield 7.1-8.0 g (80-90%)

R_(f) (1)=0.40-0.50; m.p.: 86°-88° C.

α!_(D) ²⁰ =-149° (c=1, methanol)

Analysis for C₁₀ H₁₀ O₃ (178.18) Calculated: C %=67.4; H %=5.66; Found:C %=67.4; H %=5.65.

Step B: L-Isochroman-3-carbonyl-L-proline t-butyl ester

10 4.45 g (25 mmole) of L-isochroman-3-carboxylic acid Example 4, StepA! and 5.2 g (25 mmole) of L-proline t-butyl ester hydrochloride arecondensed according to the process described in Example 1, Step B,applying identical amounts of solvents and reagents. The oily product isL-isochroman-3-carbonyl-L-proline t-butyl ester.

Yield 25 mmole.

R_(f) (1)=0.80-0.90.

Step C: L-Isochroman-3-carbonyl-L-proline cyclohexylammonium salt

The oily product of Example 4, Step B is transformed according to theprocess described in Example 3, Step C, using identical amounts ofsolvents and reagents.

Yield 6.5 g (70%)

R_(f) (1)=0.40-0.50; m.p.: 148°-150° C.

α!_(D) ²⁰ =-91.9° (c=1, methanol)

Analysis for C₂₁ H₃₀ N₂ O₄ (374.47) Calculated: C %=67.35; H %=8.07; N%=7.48; Found: C %=67.2; H %=8.0; N %=7.4.

EXAMPLE 5 3-Phenyl-D-lactyl-L-prolyl-L-arginine aldehyde hemisulfate

Step 1: O-Tetrahydropyranyl-3-phenyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

2.68 g (6 mmole) of O-tetrahydropyranyl-3-phenyl-D-lactyl-L-prolinecyclohexylammonium salt (Example 5, Step B) is dissolved in 30 ml ofdichloromethane and 7 ml of 1M potassium hydrogen sulfate. Thedichloromethane layer is washed with water, dried over sodium sulfateand evaporated at 20-25 millibar from a water bath at about 40° C.Transforming the residue and 2.73 g (7 mmole) oft-butyloxycarbonyl-N^(G) -benzyloxycarbonyl-L-arginine lactam S. Bajuszet al.: J. Med. Chem. 33, 1729 (1990)! yields 2.3 g (65%) of the aimedproduct.

R_(f) (ethyl acetate)=0.55-0.65.

Analysis for C₃₃ H₄₁ N₅ O₇ (619.70) Calculated: C %=63.95; H %=6.67; N%=11.3; Found: C %=63.8; H %=6.7; N %=11.1.

Step 2: O-Tetrahydropyranyl-3-phenyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

Transforming 2.2 g (3.5 mmole) ofO-tetrahydropyranyl-3-phenyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 5, Step 1) according tothe process described in Example 1, Step 2 by using proportional amountsof solvents and reagents, 1.30 g (60%) of the aimed product is obtained.

R_(f) (2)=0.40-0.50.

Analysis for C₃₃ H₄₃ N₅ O₇ (621.71) Calculated: C %=63.75; H %=6.97; N%=11.26; Found: C %=63.85; H %=7.1; N %=11.1.

Step 3: 3-Phenyl-D-lactyl-L-prolyl-L-arginine aldehyde hemisulfate

Transforming 1.25 g (2 mmole) ofO-tetrahydropyranyl-3-phenyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 5, Step 2) according tothe process described in Example 7, Step 3, using proportional amountsof solvents and reagents, 0.6 g (60%) of the aimed product is obtained.

R_(f) (5)=0.50-0.60; HPLC(I/1): k'=3.96, 4.96.

FAB mass spectrum (404 M+H!⁺) confirms the assumed structure.

The starting materials can be prepared as follows:

O-Tetrahydropyranyl-3-phenyl-D-lactyl-L-proline cyclohexylammonium salt

Step A: O-Tetrahydropyranyl-3-phenyl-D-lactic acid cyclohexylammoniumsalt

6.64 g (40 mmole) of 3-phenyl-D-lactic acid and 3.7 g (44 mmole) ofdihydropyran are reacted in acetonitrile according to the method ofObrecht and Heimgartner Helv. Chim. Acta 67, 526 (1984)!. The reactionmixture is evaporated at 20-25 millibar from a water bath at about 40°C. The residue is dissolved in 20 ml of dichloromethane, washed withwater, dried over sodium sulfate and repeatedly evaporated. The obtainedO-tetrahydropyranyl-3-phenyl-D-lactic acid is dissolved in diethyl etherand converted into cyclohexylammonium salt. The crystals are filteredoff, washed with diethyl ether and dried at reduced pressure.

Yield 8.4 g (60%)

R_(f) (9)=0.68-0.77; m.p.: 148°-150° C.

Analysis for C₂₀ H₃₁ NO₄ (349.46) Calculated: C %=68.74; H %=8.94; N%=4.01; Found: C %=68.65; H %=9.1; N %=4.1.

Step B: O-Tetrahydropyranyl-3-phenyl-D-lactyl-L-prolinecyclohexylammonium salt

8.74 g (25 mmole) of O-tetrahydropyranyl-3-phenyl-D-lactic acidcyclohexylammonium salt (Example 5, Step A) is dissolved in 30 ml of 1Mpotassium hydrogen sulfate and 50 ml of dichloromethane. Thedichloromethane layer is washed with water, dried over sodium sulfateand evaporated at 20-25 millibar from a water bath at about 40° C. Theobtained O-tetrahydropyranyl-3-phenyl-D-lactic acid and 4.15 g (25mmole) of L-proline methyl ester hydrochloride are condensed by themethod of Obrecht and Heimgartner Helv. Chim. Acta 67, 526 (1984)!, theO-tetrahydropyranyl-3-phenyl-D-lactyl-L-proline methyl ester formed issaponified and the resultingO-tetrahydropyranyl-3-phenyl-D-lactyl-L-proline is converted in diethylether solution to the crystalline cyclohexylammonium salt.

Yield 6.7 g (60%)

R_(f) (1)=0.3-0.4

Analysis for C₂₅ H₃₈ N₂ O₅ (446.57) Calculated: C %=67.23; H %=8.58; N%=6.27; Found: C %=67.3; H %=8.7; N %=6.1.

EXAMPLE 6 D-2-Phenyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehydehemisulfate

Step 1: O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

Starting from 2.6 g (6 mmole) ofO-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-prolinecyclohexylammonium salt (Example 6, Step B) and 2.73 g (7 mmole) oft-butyloxycarbonyl-N^(G) -benzyloxycarbonyl-L-arginine lactam S. Bajuszet al.: J. Med. Chem. 33, 1729 (1990)! and applying the processdescribed in Example 5, Step 1 in every respect, by using identicalamounts of solvents and reagents, 2.5 g (69%) of the oily product isobtained which is directly used in the subsequent step. R_(f) (ethylacetate)=0.5-0.6!.

Step 2: O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

Transforming 2.5 g (4.1 mmole) ofO-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 6, Step 1) according tothe process described in Example 5, Step 2, using proportional amountsof solvents and reagents, 1.50 g (60%) of the aimed product is obtained.

R_(f) (2)=0.33-0.44

Analysis for C₃₂ H₄₁ N₅ O₇ (607.69) Calculated: C %=63.24; H %=6.80; N%=11.52; Found: C=63.1; H %=6.9; N %=11.35.

Step 3: D-2-Phenyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehydehemisulfate

Transforming 1.22 g (2 mmole) ofO-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 6, Step 2) according tothe process described in Example 7, Step 3, using proportional amountsof solvents and reagents, 0.6 g (60%) of the aimed product is obtained.

R_(f) (5)=0.46-0.56; HPLC(I/4): k'=8.33, 9.22, 14.20.

FAB mass spectrum (390 M+H!⁺) confirms the assumed structure.

The starting materials can be prepared as follows:

O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-prolinecyclohexylammonium salt

Step A: O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetic acidcyclohexylammonium salt

Transforming 6.1 g (40 mmole) of D-mandelic acid according to theprocess described in Example 5, Step A, using identical amounts ofsolvents and reagents, 8.4 g (62%) of the aimed product is obtained.

R_(f) (9)=0.68-0.77; m.p.: 148°-150° C.

Analysis for C₁₉ H₂₉ NO₄ (335.43) Calculated: C %=68.03; H %=8.71; N%=4.18; Found: C %=67.95; H %=8.8; N %=4.1.

Step B: O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-prolinecyclohexylammonium salt

Transforming 8.4 g (25 mmole) ofO-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetic acid cyclohexylammoniumsalt (Example 6, Step A) and 4.15 g (25 mmole) of L-proline methyl esterhydrochloride according to the process described in Example 5, Step B,by using identical amounts of solvents and reagents, 6.7 g (62%) of theaimed product is obtained.

R_(f) (1)=0.3-0.4.

Analysis for C₂₄ H₃₆ N₂ O₅ (432.54) Calculated: C %=66.64; H %=8.39; N%=6.48; Found: C %=66.5; H %=8.25; N %=6.3.

EXAMPLE 7 3-Cyclohexyl-D-lactyl-L-prolyl-L-arginine aldehyde hemisulfate

Step 1: O-Tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

2.72 g (6 mmole) of O-tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-prolinecyclohexylammonium salt (Example 7, Step D) is dissolved in 30 ml ofdichloromethane and 7 ml of 1M potassium hydrogen sulfate. Thedichloromethane layer is washed with water, dried over sodium sulfateand evaporated from a water bath at about 40° C. and 20-25 millibar.Reacting the residue and 2.73 g (7 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 33,1729 (1990)! according the procedure described in Example 1, Step 1,applying identical amounts of solvents and reagents, 2.44 g (65%) of theaimed product is obtained.

R_(f) (2)=0.73-0.80

Step 2: O-Tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

2.2 g (3.5 mmole) ofO-tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 7, Step 1) is dissolved in8 ml of tetrahydrofuran, then a solution of 2.625 mmole of lithiumaluminium hydride, dissolved in tetrahydrofuran, is added at atemperature of -50° C., under constant stirring. The progress of thereduction is monitored by thin-layer chromatography in a developingsolvent system of ethyl acetate:pyridine:acetic acid:water(240:20:6:11). If required, further portions of lithium aluminiumhydride are added. The reaction mixture is acidified to pH 3 with 1Mpotassium hydrogen sulfate under cooling and stirring. The solution isdiluted with water up to the point when it turns cloudy (approx. 10 ml),washed with 2×10 ml of a mixture of n-hexane:ethyl acetate (1:1) andextracted with 3×10 ml of methylene chloride. The methylene chloridelayers are pooled, washed with 3×5 ml of water, 5% aqueous sodiumhydrogen carbonate, then again with water, dried over sodium sulfate andevaporated from a water bath at about 40° C., at 20-25 millibar. Theevaporation residue is worked up with n-hexane, filtered and dried atreduced pressure.

Yield 1.30 g (59%).

R_(f) (2)=0.26-0.38.

Analysis for C₃₃ H₄₉ N₅ O₇ (627.76) Calculated: C %=63.13; H %=7.87; N%=11.16 Found: C %=63.3; H %=8.0; N %=11.0.

Step 3: 3-Cyclohexyl-D-lactyl-L-prolyl-L-arginine aldehyde hemisulfate

1.25 g (2 mmole) ofO-tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 7, Step 2) is dissolvedin 10 ml of 60% aqueous ethanol containing 1.05 mmole of sulfuric acidand submitted to hydrogenation in the presence of a Pd--C catalyst. Theprogress of the reaction is monitored by thin-layer chromatography in adeveloping solvent system of ethyl acetate:pyridine:acetic acid:water(45:20:6:11) (R_(f) =0.45-0.54; intermediate product free frombenzyloxycarbonyl group). The reaction is completed in about 60 minutes,then the catalyst is filtered off and washed with 60% aqueous ethanoland water. The filtrate is concentrated to about half of its volume atreduced pressure, diluted with water to 10 ml, its pH is adjusted to 2.8with sulfuric acid or an anion-exchange resin (HO⁻) and then it is leftto stand at room temperature for about 24 hours till the completecleavage of the tetrahydropyranyl protecting group (reaction ismonitored by thin-layer chromatography in the above solvent system). Theaqueous solution is washed with 3×5 ml of methylene chloride, the pH isadjusted to 3.6 with an anion-exchange resin (HO⁻), then the solution isfrozen and freeze-dried.

Yield 0.6 g (60%)

R_(f) (5)=0.43-0.51; HPLC(I/3): k'=35.25 and 37.40

FAB mass spectrum (410 M+H!⁺) confirms the assumed structure.

The starting materials can be prepared as follows:

O-Tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-proline cyclohexylammoniumsalt

Step A: 3-Cyclohexyl-D-lactic acid

5 g (30.1 mmole) of 3-phenyl-D-lactic acid R_(f) (2)=0.45-0.55! isdissolved in 70 ml of acetic acid and submitted to hydrogenation in thepresence of 0.25 g of platinum(IV)oxide catalyst in the temperaturerange of 40° to 50° C. The reaction is monitored by thin-layerchromatography final product: R_(f) (2)=0.6-0.7!. After completedreaction the catalyst is filtered off, the filtrate is evaporated from awater bath at a temperature not higher than 40° C., at 20-25 millibar.Then 20 ml of toluene is evaporated twice from the residue which isfinally recrystallized from n-hexane.

Yield 4.3 g (83%)

R_(f) (2)=0.60-0.70; m.p.: 90°-93° C.

α!_(D) ²⁰ =+8.45° (c=1, methanol)

Analysis for C₉ H₁₆ O₃ (172.22) Calculated: C %=62.76; H %=9.36; Found:C %=62.65; H %=9.5;

Step B: 3-Cyclohexyl-D-lactic acid methyl ester

3.44 g (20 mmole) of 3-cyclohexyl-D-lactic acid (Example 7, Step A)R_(f) (9)=0.40-0.45! is dissolved in 30 ml of anhydrous methanol, 4drops of concentrated sulfuric acid are added, and the mixture isrefluxed for 3 hours. The solution is evaporated from a water bath at atemperature not exceeding 40° C., at 20-25 millibar, the residue isdissolved in 40 ml of methylene chloride and washed with water toneutrality. The methylene chloride solution is dried over sodium sulfateand evaporated under conditions described above. In this way 3.8 g (20mmole) of 3-cyclohexyl-D-lactic acid methyl ester R_(f) (9)=0.80-0.86!is obtained as an oil.

Step C: O-Tetrahydropyranyl-3-cyclohexyl-D-lactic acidcyclohexylammonium salt

20 mmole of 3-cyclohexyl-D-lactic acid methyl ester (Example 7, Step B)is dissolved in 20 ml of methylene chloride, then 2 ml (22 mmole) ofdihydropyran and 0.3 ml of ethyl acetate containing hydrogen chloride(concentration: 0.11-0.15 g/ml) are added at constant stirring. Thesolution is left to stand at room temperature overnight, thereafter itis diluted with 20 ml of methylene chloride, washed with water toneutrality and evaporated from a water bath at a temperature notexceeding 40° C., at 20-25 millibar. The resulting oil 4.4 g ofO-tetrahydropyranyl-3-cyclohexyl-D-lactic acid methyl ester, R_(f)(9)=0.84-0.90! is dissolved in 40 ml of methanol, then 20 ml of 1Msodium hydroxide is added. The reaction mixture is left to stand at roomtemperature overnight, thereafter 20 ml of water is added and thesolution is concentrated from a water bath at a temperature not higherthan 40° C., at 20-25 millibar up to the appearance of mild cloudiness.The solution obtained is washed with 10 ml of methylene chloride, cooledto 5°-10° C., the pH is adjusted to 3 with 1M potassium hydrogen sulfateand extracted with 3×10 ml of methylene chloride. The methylene chloridelayers are pooled, washed with water to neutrality, dried over sodiumsulfate and evaporated under conditions mentioned above. TheO-tetrahydropyranyl-3-cyclohexyl-D-lactic acid obtained is dissolved indiethyl ether and converted to cyclohexylammonium salt. The crystals arefiltered off, washed with diethyl ether and dried at reduced pressure.

Yield 5.0 g (70%)

M.p.: 153°-155° C. (sintering at 142° C.).

R_(f) (9)=0.63-0.70;

Analysis for C₂₀ H₃₇ NO₄ (355.50) Calculated: C %=67.57; H %=10.49; N%=3.98; Found: C %=68.0; H %=11.0; N %=3.85.

Step D: O-Tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-prolinecyclohexylammonium salt

4.45 g (12.5 mmole) of O-tetrahydropyranyl-3-cyclohexyl-D-lactic acidcyclohexylammonium salt (Example 7, Step C) is dissolved in 15 ml of a1M solution of potassium hydrogen sulfate and 25 ml of methylenechloride. The methylene chloride layer is washed with water, dried oversodium sulfate and evaporated from a water bath at a temperature notexceeding 40° C., at 20-25 millibar. The resultingO-tetrahydropyranyl-3-cyclohexyl-D-lactic acid is dissolved in 15 ml ofdimethyl formamide, the solution is cooled in ice water, then 2.06 g(12.5 mmole) of L-proline methyl ester hydrochloride, 2.10 g (13.75mmole) of 1-hydroxybenzotriazole hydrate, 1.75 ml of triethylamine (12.5mmole) and 2.58 g (12.5 mmole) of dicyclohexylcarbodiimide are added.The reaction mixture is left to stand overnight at room temperature, theprecipitated dicyclohexylurea is filtered and the filtrate is evaporatedfrom a water bath at a temperature not exceeding 40° C., at 20-25millibar. The residue is dissolved in 30 ml of methylene chloride,washed with 10 ml of water, 2×10 ml of 5% sodium hydrogen carbonatesolution and again with water, dried over sodium sulfate and evaporatedfrom a water bath at about 40° C. and 20-25 millibar. The resulting oilO-tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-proline methyl ester, R_(f)(9)=0.86-0.93! is dissolved in 40 ml of methanol, 13 ml of a solution of1M sodium hydroxide is added and the mixture is stirred for 24 hours.Thereafter 15 ml of a 1M sodium hydroxide solution and 20 ml ofmethylene chloride are added. The two layers formed are separated, theaqueous phase is acidified to pH 3 with 1M potassium hydrogen sulfateunder constant stirring and ice cooling and extracted with 2×15 ml ofmethylene chloride. The methylene chloride layers are pooled, washedwith water to neutrality, dried over sodium sulfate and evaporated underconditions described above. The resultingO-tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-proline is converted to thecrystalline cyclohexylammonium salt in diethyl ether solution.

Yield 3.2 g (56%); m.p.: 119°-120° C.

R_(f) (9)=0.44-0.51

Analysis for C₂₅ H₄₄ N₂ O₅ (452.62) Calculated: C %=66.34; H %=9.80; N%=6.19; Found: C %=66.4; H %=9.9; N %=6.25.

EXAMPLE 8 D-2-Cyclohexyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehydehemisulfate

Step 1:O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

Starting from 2.6 g (6 mmole) ofO-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolinecyclohexylammonium salt (Example 8, Step C) and 2.73 g (7 mmole) oft-butyloxycarbonyl-N^(G) -benzyloxycarbonyl-L-argninine lactam S. Bajuszet al.: J. Med. Chem. 33, 1729 (1990)! the procedure described inExample 5, Step 1 is applied, using identical amounts of solvents andreagents.

Yield: 2.53 g (69%)

R_(f) (2)=0.70-0.78.

Step 2:O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

2.15 g (3.5 mmole) ofO-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 8, Step 1) is transformedaccording to the process described in Example 7, Step 2, applyingidentical amounts of solvents and reagents.

Yield: 1.40 g (65%)

R_(f) (2)=0.23-0.31

Analysis for C₃₂ H₄₇ N₅ O₇ (613.74) Calculated: C %=62.62; H %=7.72; N%=11.41 Found: C %=62.8; H %=7.8; N %=11.2.

Step 3: D-2-Cyclohexyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehydehemisulfate

1.23 g (2 mmole) ofO-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 8, Step 2) istransformed according to the process described in Example 7, Step 3,applying identical amounts of solvents and reagents.

Yield: 0.6 g (60%)

R_(f) (5)=0.39-0.46.

HPLC(I/3): k'=5.70, 6.10 and 8.76.

FAB mass spectrum confirms the assumed structure (396 M+H!⁺).

The starting material can be prepared as follows:

O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolinecyclohexylammonium salt

Step A: D-2-Cyclohexyl-2-hydroxyacetic acid methyl ester

Starting from 3.2 g (20 mmole) of D-2-cyclohexyl-2-hydroxyacetic acid H.Lettre, H. Barnback and H. Staunau: Chem Ber. 69, 1594-1598 (1936) R_(f)(9)=0.32-0.42! and following the process described in Example 7, Step B,applying identical amounts of solvents and reagents, 3.5 g (20 mmole) ofan oily product R_(f) (9)=0.78-0.84!, considered to beD-2-cyclohexyl-2-hydroxyacetic acid methyl ester, is obtained.

Step B: O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetic acidcyclohexylammonium salt

Starting from 20 mmole of D-2-cyclohexyl-2-hydroxyacetic acid methylester (Example 8, Step A) and following the process described in Example7, Step C, applying identical amounts of solvents and reagents, 4.1 g ofan oily intermediary productO-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetic acid methyl esterR_(f) (9)=0.80-0.88!, then the aimed product is obtained.

Yield: 4.1 g (60%)

R_(f) (9)=0.55-0.70

M.p.: 152°-155° C. (sintering at 122° C.)

Analysis for C₁₉ H₃₅ NO₄ (341.48) Calculated: C %=66.82; H %=10.33; N%=4.10; Found: C %=66.9; H %=10.3; N %=4.2.

Step C: O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-prolinecyclohexylammonium salt

Starting from 4.3 g (12.5 mmole) ofO-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetic acidcyclohexylammonium salt (Example 8, Step B) and 2.1 g (12.5 mmole) ofL-proline methyl ester hydrochloride and proceeding according to theprocess described in Example 7, Step D, applying identical amounts ofsolvents and reagents, the oily intermediary productO-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-proline methylester R_(f) (9)=0.84-0.93!, then the aimed product is obtained.

Yield: 3.25 g (60%)

R_(f) (9)=0.20-0.27; m.p.: 140°-142° C.

Analysis for C₂₄ H₄₂ N₂ O₅ (438.61) Calculated: C %=65.72; H %=9.65; N%=6.39; Found: C %=65.7; H %=9.7; N %=6.3.

EXAMPLE 9 3-Cyclohexyl-D-lactyl-L-pipecolyl-L-arginine aldehydehemisulfate

Step 1: O-Tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

3.9 g (10 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 33,1729 (1990)) is suspended in 15 ml of chloroform, then 14.5 ml of ethylacetate containing hydrogen chloride gas (concentration 0.11-0.15 g/ml)are added under stirring and cooling in ice water. After two hours thereaction mixture is diluted with 15-20 ml of diethyl ether, the crystalsformed are filtered off, washed with 7 ml of acetone and 7 ml of diethylether and dried over potassium hydroxide at reduced pressure overnight.The thus-obtained N^(G) -benzyloxycarbonyl-L-arginine lactamhydrochloride is dissolved in 10 ml of pyridine, cooled to -15° C., thenat this temperature a solution of 3.7 g (10 mmole) ofO-tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-pipecolic acid (Example 9,Step A) in 10 ml of pyridine, 0.964 ml (10 mmole) of phosphorusoxychloride and 1.85 ml (13.2 mmole) of triethylamine are added. Thereaction mixture is stirred for 30 minutes at -15° C. and for one hourat 0° C., then it is evaporated from a water bath at a temperature ofabout 40° C. at 20-25 millibar. The residue is dissolved in 30 ml ofwater and 30 ml of ethyl acetate, the ethyl acetate layer is washed with10 ml portions each of water, 5% sodium hydrogen carbonate solution,water, 1M potassium hydrogen sulfate solution and again water, driedover sodium sulfate, then evaporated from a water bath at about 40° C.at 20-25 millibar. The oily product is submitted to chromatography on asilicagel adsorbent in a developing system of ethylacetate:pyridine:acetic acid:water (960:20:6:11). The fractionscontaining the pure product are pooled, extracted with 2×10 ml of 1Mpotassium hydrogen sulfate solution, washed with water to neutrality andevaporated to dryness at reduced pressure as mentioned above.

Yield: 2.56 g (40%) oil

R_(f) (2)=0.83-0.90

Step 2: O-Tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

2.25 g (3.5 mmole) ofO-tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 9, Step 1) is transformedaccording to the process described in Example 7, Step 2, applyingidentical amounts of solvents and reagents.

Yield: 1.46 g (65%)

R_(f) (2)=0.46-0.58

Analysis for C₃₄ H₅₁ N₅ O₇ (641.79) Calculated: C %=63.62; H %=8.01; N%=10.91; Found: C %=63.80; H %=8.15; N %=10.9.

Step 3: 3-Cyclohexyl-D-lactyl-L-pipecolyl-L-arginine aldehydehemisulfate

1.3 g (2 mmole) ofO-tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 9, Step 2) istransformed according to the process described in Example 7, Step 3,applying identical amounts of solvents and reagents.

Yield: 0.6 g (60%)

R_(f) (5)=0.51-0.60

HPLC(I/3): k'=7.34, 7.97, 8.18, 8.45, 8.55, 8.90

The FAB mass spectrum confirms the assumed structure (424 M+H!⁺).

The starting materials can be prepared as follows:

Step A: O-Tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-pipecolic acid

4.45 g (12.5 mmole) of O-tetrahydropyranyl-3-cyclohexyl-D-lactic acidcyclohexylammonium salt (Example 7, Step C) is dissolved in 15 ml of 1Mpotassium hydrogen sulfate and 25 ml of methylene chloride. Themethylene chloride layer is washed with water, dried over sodium sulfateand evaporated from a water bath at about 40° C. and 20-25 millibar. Theresulting O-tetrahydropyranyl-3-cyclohexyl-D-lactic acid is dissolved in15 ml of dimethyl formamide, cooled in an ice bath, then 2.24 g (12.5mmole) of L-pipecolic acid methyl ester hydrochloride, 2.10 g (13.75mmole) of 1-hydroxybenzotriazole hydrate, 1.75 ml (12.5 mmole) oftriethylamine and 2.58 g (12.5 mmole) of dicyclohexylcarbodiimide areadded. The reaction mixture is left to stand at room temperatureovernight, the precipitated dicyclohexylurea is filtered off and thesolution is evaporated from a water bath of about 4.0C at 20-25millibar. The residue is dissolved in 30 ml of methylene chloride,washed with 10 ml each of water, 2×10 ml of 5% sodium hydrogen carbonateand again with water, dried over sodium sulfate, then evaporated from awater bath of about 40° C. at 20-25 millibar. The oily residueO-tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-pipecolic acid methyl ester;R_(f) (9)=0.95-0.98! is dissolved in 40 ml of methanol, then 13 ml of 1Msodium hydroxide solution is added and the solution is stirred for 24hours. Thereafter 15 ml of 1M sodium hydroxide solution and 20 ml ofmethylene chloride are added. The two layers obtained are separated, thepH of the aqueous layer is adjusted to 3 with 1M potassium hydrogensulfate under stirring and ice cooling, then extracted with 2×15 ml ofmethylene chloride. The methylene chloride solutions are pooled, washedto neutrality with water, dried over sodium sulfate and evaporated asdescribed above, yielding 3.75 g (82%) ofO-tetrahydropyranyl-3-cyclohexyl-D-lactyl-L-pipecolic acid in amorphousform.

R_(f) (9)=0.51-0.63

Analysis for C₂₀ H₃₃ NO₅ (367.47) Calculated: C %=65.36; H %=9.05; N%=3.81 Found: C %=63.35; H %=9.2; N %=4.0

EXAMPLE 10 D-2-Cyclohexyl-2-hydroxyacetyl-L-pipecolyl-L-argininealdehyde hemisulfate

Step 1:O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

Starting from 3.55 g (10 mmole) ofO-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecolic acid(Example 10, Step A) and 3.9 g (10 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 33,1729 (1990)! and proceeding according to the process described inExample 9, Step 1, applying identical amounts of solvents and reagents,2.8 g (45%) of the aimed product is obtained.

R_(f) (2)=0.80-0.88

Step 2: O-Tetrahydropyranyl-D-2-cyclohexyl-²-hydroxyacetyl-L-pipecolyl-N^(G) -benzyloxycarbonyl-L-arginine aldehyde

2.2 g (3.5 mmole) ofO-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 10, Step 1) is transformedaccording to the process described in Example 9, Step 2, applyingidentical amounts of solvents and reagents.

Yield 1.45 g (66%)

R_(f) (2)=0.42-0.53

Analysis for C₃₃ H₄₉ N₅ O₇ (627.76) Calculated: C %=63.13; H %=7.87; N%=11.16; Found: C %=63.3; H %=7.95; N %=11.0.

Step 3: D-2-Cyclohexyl-2-hydroxyacetyl-L-pipecolyl-L-arginine aldehydehemisulfate

1.26 g (2 mmole) ofO-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 10, Step 2) istransformed according to the process described in Example 7, Step 3,applying identical amounts of solvents and reagents.

Yield 0.62 g (65%)

R_(f) (5)=0.50-0.58

HPLC(I/3): k'=6.16, 7.17 and 7.67.

The FAB mass spectrum confirms the assumed structure (410 M+H!⁺).

The starting material can be prepared as follows:

Step A: O-Tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetyl-L-pipecolicacid

Starting from 4.27 g (12.5 mmole) ofO-tetrahydropyranyl-D-2-cyclohexyl-2-hydroxyacetic acidcyclohexylammonium salt (Example 8, Step B) and 2.47 g (13.75 mmole) ofL-pipecolic acid methyl ester hydrochloride, and proceeding according tothe process described in Example 9, Step A, applying identical amountsof solvents and reagents, 3.7 g (84%) of the aimed product is obtainedin amorphous form.

R_(f) (9)=0.50-0.60

Analysis for C₁₉ H₃₁ NO₅ (353.45) Calculated: C %=64.56; H %=8.84; N%=3.96; Found: C %=66.65; H %=8.85; N %=4.05.

EXAMPLE 11 D-2-Phenyl-2-hydroxyacetyl-L-pipecolyl-L-arginine aldehydehemisulfate

Step 1: O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

Starting from 3.47 g (10 mmole) ofO-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-pipecolic acid (Example11, Step A) and 3.9 g (10 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 33,1729 (1990)! and proceeding according to the process described inExample 9, Step 1, applying identical amounts of solvents and reagents,2.54 g (41%) of the aimed product is obtained.

R_(f) (2)=0.80-0.85

Step 2: O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

2.2 g (3.5 mmole) ofO-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 11, Step 1) is transformedaccording to the process described in Example 9, Step 2, applyingidentical amounts of solvents and reagents.

Yield 1.3 g (65%)

R_(f) (2)=0.40-0.45

Step 3: D-2-Phenyl-2-hydroxyacetyl-L-pipecolyl-L-arginine aldehydehemisulfate

1.24 g (2 mmole) ofO-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-pipecolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 11, Step 2) istransformed according to the process described in Example 7, Step 3,applying identical amounts of solvents and reagents.

Yield 0.56 g (60%)

R_(f) (5)=0.48-0.53

HPLC(I/2): k'=4.55, 5.78 and 10.78.

The FAB mass spectrum confirms the assumed structure (404 M+H!⁺).

The starting material can be prepared as follows:

Step A: O-Tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl-L-pipecolic acid

Starting from 4.2 g (12.5 mmole) ofO-tetrahydropyranyl-D-2-phenyl-2-hydroxyacetyl cyclohexylammonium salt(Example 6, Step A) and 2.47 g (13.75 mmole) of L-pipecolic acid methylester hydrochloride and proceeding according to the process described inExample 9, Step A, applying identical amounts of solvents and reagents,3.65 g (84%) of the aimed product is obtained in amorphous form.

R_(f) (9)=0.43-0.53

Analysis for C₁₉ H₂₅ NO₅ (347.40) Calculated: C %=65.69; H %=7.25; N%=4.03; Found: C %=65.8; H %=7.4; N %=4.3.

EXAMPLE 12 3,3-Diphenyl-D-lactyl-L-prolyl-L-arginine aldehydehemisulfate

Step 1: O-Tetrahydropyranyl-3,3-diphenyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

2.73 g (7 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 33,1729 (1990)! is suspended in 10 ml of chloroform, then 10 ml of ethylacetate containing hydrogen chloride gas (concentration: 0.11-0.15 g/ml)is added under stirring and cooling in ice water. After two hours thereaction mixture is diluted with 10-15 ml of diethyl ether, the crystalsformed are filtered, washed with 5 ml of acetone and 5 ml of diethylether and dried over potassium hydroxide at reduced pressure overnight.The N^(G) -benzyloxycarbonyl-L-arginine lactam hydrochloride obtained isdissolved in 10 ml of dimethyl formamide, cooled to -20° C. and added tothe mixed anhydride to be prepared as follows:

2.60 g (6 mmole) of O-tetrahydropyranyl-3,3-diphenyl-DL-lactyl-L-prolinehemihydrate (Example 12, Step E) is dissolved in 6 ml of dimethylformamide, refrigerated to -20° C., then under stirring 0.66 ml (6mmole) of N-methylmorpholine and 0.79 ml of isobutyl chloroformate areadded. Stirring is continued for 10 minutes, then the above suspensionof N^(G) -benzyloxycarbonyl-L-arginine lactam in dimethyl formamide andfinally 2.1 ml (15 mmole) of triethylamine are added. Stirring iscontinued for 2 hours under cooling, then the mixture is left to warm upto room temperature. Thereafter the salts are filtered off and thefiltrate is diluted with 40 ml of methylene chloride. The solutionobtained is washed with 10 ml of 1M potassium hydrogen sulfate, 3×10 mlof water, dried over sodium sulfate and evaporated from a water bath atabout 30° C. at 20-25 millibar. The resulting 3.8 g (90%) of theproduct--a mixture of compounds containing 3,3-diphenyl-D and L-lacticacid residues--is submitted to chromatography on a silicagel column,applying a mixture of ethyl acetate and diisopropyl ether (7:3) asdeveloping solvent (R_(f) =0.23-0.27 and 0.28-0.32 in this system). Thefractions containing the pure D product, migrating with higher R_(f)value, and those containing the pure L product, migrating with lowerR_(f) value, are combined, evaporated from a water bath at about 30° C.and 20-25 millibar and finally worked up with diisopropyl ether. Yield1.0 g (48%) of D and 0.98 g (47%) of L product. Product D is the peptidelactam containing 3,3-diphenyl-D-lactic acid residue. The aldehydederivative with high antithrombin activity is prepared from this productby the process described in Steps 2 and 3 of this Example.

R_(f) (10)=0.28-0.32

M.p.: 156° C. (sintering at 150° C.)

Analysis for C₃₉ H₄₅ N₅ O₇ (695.79) Calculated: C %=67.32; H %=6.52; N%=10.07; Found: C %=67.4; H %=6.65; N %=10.0.

Step 2: O-Tetrahydropyranyl-3,3-diphenyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

0.95 g (1.36 mmole) ofO-tetrahydropyranyl-3,3-diphenyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 12, Step 1) is dissolvedin 8 ml of tetrahydrofuran, then under stirring and at a temperaturebelow -20° C. 1.02 mmole of lithium aluminium hydride--dissolved intetrahydrofuran--is added. The progress of the reduction is monitored bythin-layer chromatography, applying ethyl acetate:pyridine:aceticacid:water (240:20:6:11) as developing system. If required, furtherportions of lithium aluminium hydride are added, then cool 0.5M sulfuricacid is added dropwise to the reaction mixture under cooling andstirring, adjusting the pH to 3. The solution obtained is extracted with5 ml of diethyl ether. To the bottom layer 5% sodium hydrogen carbonatesolution is added, adjusting the pH to about 6.5, then the solution isextracted with 3×5 ml of methylene chloride. The methylene chloridesolutions are pooled, dried over sodium sulfate and evaporated from awater bath at about 40° C. at 20-25 millibar. The residue is worked upwith diisopropyl ether, filtered and dried at reduced pressure.

Yield 0.86 g (90%)

R_(f) (2)=0.38-0.42

Step 3: 3,3-Diphenyl-D-lactyl-L-prolyl-L-arginine aldehyde hemisulfate

0.8 g (1.15 mmole) ofO-tetrahydropyranyl-3,3-diphenyl-D-lactyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 12, Step 2) R_(f)(4)=0.80-0.85! is dissolved in a mixture of 6 ml of dimethyl formamideand 1.2 ml of 0.5M sulfuric acid, 0.12 g of Pd--C catalyst is added andthe mixture is submitted to hydrogenation at about 10° C. The progressof the reaction is monitored by thin-layer chromatography. Theintermediary product--devoid of benzyloxycarbonyl group--has R_(f)(4)=0.43-0.47. The reaction is completed in about 30 minutes, thecatalyst is filtered off, washed with 1 ml of 50% aqueous dimethylformamide and 1 ml of water, then the combined solutions are evaporatedfrom a water bath at about 40° C. and 20-25 millibar. The oily residueis dissolved in 10 ml of water, the pH of the solution is adjusted to2.8 with sulfuric acid or an anion-exchange resin (HO⁻) and the solutionis left to stand at room temperature for about 24 hours up to the fulldeblocking of the O-tetrahydropyranyl protecting group R_(f)(4)=0.40-0.50!. The aqueous solution is washed with 3×5 ml of methylenechloride, the pH is adjusted with an anion-exchange resin (HO⁻) to 3.6,then the solution is frozen and freeze-dried.

Yield 0.5 g (70%)

R_(f) (5)=0.48-0.52

HPLC(I/3): k'=7.8 and 8.23

The FAB mass spectrum confirms the assumed structure (480 M+H!⁺).

The starting materials can be prepared as follows:

O-Tetrahydropyranyl-3,3-diphenyl-DL-lactyl-L-proline

Step A: 3,3-Diphenyl-DL-lactic acid methyl ester

22.33 g (0.1 mole) of 3,3-diphenyl-DL-lactic acid nitrile Wiese: Ann.248, 39-41 (1888)! is dissolved in 100 ml of diethyl ether, and then4.04 ml (0.1 mole) of methanol is added. The solution is saturated withdry hydrogen chloride gas under ice cooling, then it is left to standfor one hour and evaporated from a water bath at about 40° C. at 20-25millibar. The residue is crystallized with about 200 ml of diethylether, the crystals are filtered off, washed with diethyl ether anddried at reduced pressure. The product obtained 20.55 g (70%),3,3-diphenyl-DL-lactiminomethyl ether hydrochloride, (m.p.: 82°-83° C.)!is suspended in 140 ml of water and heated to 50°-60° C. for 1-2 hours.An oily product is formed which solidifies under cooling and stirring.The solid product is filtered, washed with water and then with n-hexaneand finally dried at reduced pressure.

Yield 12.8 g (50%) (calculated for the nitrile).

R_(f) (12)=0.4; m.p.: 49°-50° C.

Analysis for C₁₆ H₁₆ O₃ (256.29) Calculated: C %=74.98; H %=6.29 Found:C %=75.0; H %=6.3.

Step B: O-Tetrahydropyranyl-3,3-diphenyl-DL-lactic acid methyl ester

7.83 g (30.5 mmole) of 3,3-diphenyl-DL-lactic acid methyl ester (Example12, Step A) is dissolved in 75 ml of methylene chloride, then understirring and ice cooling 3.83 ml of dihydropyran (40 mmole) and 0.8 mlof ethyl acetate, containing hydrogen chloride gas (concentration:0.11-0.15 g/ml) are added. The solution is left to stand overnight, thenit is diluted with 50 ml of methylene chloride, washed to neutralitywith water, dried over calcium chloride and evaporated from a water bathat about 30° C. and 20-25 millibar. The residue is 30.5 mmole ofO-tetrahydropyranyl-3,3-diphenyl-DL-lactic acid methyl ester and is usedin this form in Step C.

Step C: O-Tetrahydropyranyl-3,3-diphenyl-DL-lactic acid hydrate

30.5 mmole of O-tetrahydropyranyl-3,3-diphenyl-DL-lactic acid methylester (Example 12, Step B) is dissolved in 30.5 ml of acetone and 30.5ml of ethanol containing 1M of potassium hydroxide. The progress of thereaction is monitored by thin-layer chromatography. The solution isevaporated from a water bath at about 40° C. and 20-25 millibar. Theresidue is dissolved in 40 ml of water, washed with 2×10 ml of diethylether, then acidified to pH 3 with 1M potassium hydrogen sulfate underice cooling. The separating solid product is filtered off, washed withwater and dried at reduced pressure.

Yield 7.9 g (75%); m.p.: 115°-117° C. (sintering at 76° C.)

R_(f) (9)=0.65-0.75

Analysis for C₂₀ H₂₂ O₄.H₂ O (344.39) Calculated: C %=69.75; H %=7.02;Found: C %=69.7; H %=6.55.

Step D: O-Tetrahydropyranyl-3,3-diphenyl-DL-lactic acid2,4,5-trichlorophenyl ester

4.5 g (13 mmole) of O-tetrahydropyranyl-3,3-diphenyl-DL-lactic acidhydrate (Example 12, Step C) and 2.8 g (14.2 mmole) of2,4,5-trichlorophenol are dissolved in 15 ml of tetrahydrofuran, thenunder stirring and ice cooling 2.8 g (13.6 mmole) ofdicyclohexylcarbodiimide is added. The reaction mixture is left to standat room temperature for 3 hours, the precipitated dicyclohexylurea isfiltered off and washed with tetrahydrofuran. The tetrahydrofuransolutions are combined and evaporated from a water bath at about 40° C.and 20-25 millibar. The residue is worked up with 15 ml of diisopropylether. The suspension is cooled with ice water, 5 ml of n-hexan isadded, then the suspension is filtered. The solid product is washed witha mixture of diisopropyl ether and n-hexane (1:1) and dried at reducedpressure. The product obtained isO-tetrahydropyranyl-3,3-diphenyl-DL-lactic acid 2,4,5-trichlorophenylester 4.6 g (10 mmole), R_(f) (11)=0.75-0.85!.

Step E: O-Tetrahydropyranyl-3,3-diphenyl-DL-lactyl-L-proline hemihydrate

10 mmole of O-tetrahydropyranyl-3,3-diphenyl-DL-lactic acid2,4,5-trichlorophenyl ester (Example 12, Step D) is dissolved in 10 mlof pyridine, then 1.15 g (10 mmole) of L-proline and 1.4 ml (10 mmole)of triethylamine are added. The reaction mixture is stirred at roomtemperature for 6 hours, then evaporated from a water bath at about 40°C. and 20-25 millibar. The residue is dissolved in 20 ml of water and 10ml of diethyl ether. The aqueous layer is washed with 10 ml of diethylether, acidified with 1M potassium hydrogen sulfate to pH 3 under icecooling and extracted with 3×10 ml of diethyl ether. The combineddiethyl ether solutions are washed with water, dried over sodium sulfateand evaporated as described above. The residue is worked up with 10 mlof diisopropyl ether, the suspension is cooled with ice water, thenfiltered, washed with cold diisopropyl ether and dried at reducedpressure.

Yield 3.05 g (70%)

R_(f) (2)=0.45-0.55; m.p.: 132°-135° C.

Analysis for C₂₅ H₂₉ NO₅.1/2H₂ O (432.50) Calculated: C %=69.42; H%=6.99; N %=3.24; Found: C %=69.4; H %=6.85; N %=3.2.

EXAMPLE 13 D-2-(2-Naphthyl)-2-hydroxyacetyl-L-prolyl-L-arginine aldehydehemisulfate

Step 1: O-t-Butyl-D-2-(2-naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

3.12 g (8 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 33,1729 (1990)! is suspended in 12 ml of chloroform, then under stirringand ice cooling 12 ml of ethyl acetate containing hydrogen chloride gas(concentration: 0.11-0.15 g/ml) is added. After two hours the reactionmixture is diluted with 15 ml of diethyl ether, the precipitatedcrystalline product is filtered off, washed with 6 ml of acetone and 6ml of diethyl ether and dried over potassium hydroxide under reducedpressure overnight. The obtained N^(G) -benzyloxycarbonyl-L-argininelactam hydrochloride is dissolved in 12 ml of dimethyl formamide,refrigerated to -20° C. and added to the mixed anhydride to be preparedas follows: 2.8 g (7.8 mmole) ofO-t-butyl-D-2-(2-naphthyl)-2-hydroxyacetyl-L-proline (Example 13, StepF) is dissolved in 8 ml of dimethyl formamide, then under stirring andat -20° C. 0.87 ml (7.8 mmole) of N-methylmorpholine, 1.03 ml ofisobutyl chloroformate, then after 10 minutes the above suspension ofN^(G) -benzyloxycarbonyl-L-arginine lactam in dimethyl formamide,finally 2.8 ml (20 mmole) of triethylamine are added. The reactionmixture is stirred under cooling for 2 hours, then left to warm up toroom temperature. Thereafter the salts are filtered off and the filtrateis diluted with 50 ml of benzene. The solution obtained is washed with15 ml of 1M potassium hydrogen sulfate solution and 3×15 ml of water,dried over sodium sulfate and evaporated from a water bath at about 40°C. and 20-25 millibar. The residue is worked up with n-hexane, filtered,washed with n-hexane and dried at reduced pressure.

Yield 4.14 g (85%)

R_(f) (9)=0.72-0.75

Step 2: D-2-(2-Naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

4.08 g (6.5 mmole) ofO-t-butyl-D-2-(2-naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 13, Step 1) is dissolvedin 20 ml of a mixture of trifluoroacetic acid and methylene chloride(1:1) and left to stand at room temperature for 30 minutes. The reactionmixture is diluted with 40 ml of methylene chloride, washed toneutrality with 3×20 ml of water and 20 ml of 5% sodium hydrogencarbonate, dried over sodium sulfate and evaporated from a water bath atabout 40° C. and 20-25 millibar. The residue is submitted tochromatography on a column prepared from 100 g of silicagel in adeveloping system of ethyl acetate: pyridine:acetic acid:water(480:20:6:11). The fractions containing the pure product migrating withR_(f) (7)=0.44-0.46 are pooled, washed with 1M potassium hydrogensulfate solution and water, dried over sodium sulfate and evaporatedfrom a water bath at about 40° C. and 20-25 millibar. The residue isworked up with n-hexane, filtered, washed with n-hexane and dried atreduced pressure.

Yield 1.8 g (48%)

R_(f) (7)=0.44-0.46

Analysis for C₃₁ H₃₃ N₅ O₆ (571.14) Calculated: C %=65.12; H %=5.82; N%=12.25; Found: C %=65.2; H %=5.9; N %=12.2.

Step 3: D-2-(2-Naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

1.71 g (3 mmole) of D-2-(2-naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 13, Step 2) is dissolvedin 15 ml of tetrahydrofuran, then under stirring and at a temperaturenot exceeding -20° C. a solution of 2.25 mmole of lithium aluminiumhydride in tetrahydrofuran is added. The progress of the reaction ismonitored by thin-layer chromatography in a solvent system of ethylacetate:pyridine:acetic acid:water (30:20:6:11) and, if required,further portions of lithium aluminium hydride are added. Thereafter cool0.5M sulfuric acid is added under stirring and cooling to ensure a pHvalue of 3-4 in the reaction mixture. The resulting solution is dilutedwith 15 ml of water, washed with 10 ml of ethyl acetate and extractedwith 3×10 ml of n-butanol saturated with water. The n-butanol solutionsare combined, washed with 10 ml of 5% sodium hydrogen carbonate solutionand 2×10 ml of water saturated with n-butanol, then evaporated from awater bath at about 40° C. and 20-25 millibar. The residue is worked upwith n-hexane, filtered, washed with n-hexane and dried at reducedpressure.

Yield 1.13 g (65.7%)

R_(f) (6)=0.62-0.66

Step 4: D-2-(2-Naphthyl)-2-hydroxyacetyl-L-prolyl-L-arginine aldehydehemisulfate

1.0 g (1.75 mmole) of D-2-(2-naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 13, Step 3) is dissolvedin 20 ml of ethanol, 1.75 ml of 0.5M sulfuric acid and 2 ml of water areadded and the solution is submitted to hydrogenation in the presence ofPd--C catalyst. The reaction is monitored by thin-layer chromatographyin the solvent system ethyl acetate:pyridine:acetic acid:water(30:20:6:11). The reaction is completed within about 4 hours, then thecatalyst is filtered off, washed with 5 ml of 60% aqueous ethanol and 5ml of water. The ethanol is distilled off from the filtrate underreduced pressure, then 15 ml of water is added, the solution is washedwith 10 ml of methylene chloride and its pH is adjusted to 3.3 withsulfuric acid or anion-exchange resin (HO⁻). If the solution obtained isnot entirely clear (cloudy due to precipitated material), theprecipitated material is dissolved by adding 0.5-1.5 ml of t-butanol(max. 10%), the clear solution is frozen and freeze-dried.

Yield 0.78 g (88%)

R_(f) (5)=0.29-0.32

HPLC(I/1): k'=8.25, 9.25 and 11.3.

The FAB mass spectrum confirms the assumed structure (440 M+H!⁺).

The starting material can be prepared as follows:

O-t-Butyl-D-2-(2-naphthyl)-2-hydroxyacetyl-L-proline

Step A: DL-2-(2-naphthyl)-2-hydroxyacetic acid methyl ester

Starting from 20.2 g (100 mmole) of DL-2-(2-naphthyl)-2-hydroxyaceticacid I. M. Panaiotov: Compt. rend. acad. bulgare sci. 10, No. 2, 137-140(1957); Chem. Abstr. 52, 5336e (1958); m.p.: 158°-160° C.; R_(f)(8)=0.28-0.30! and proceeding according to the process described inExample 7, Step B, applying identical amounts of solvents and reagents,22 g of an oily product is obtained R_(f) (2)=0.79-0.81! which isconsidered 100 mmole of DL-2-(2-naphthyl)-2-hydroxyacetic acid methylester.

Step B: O-t-Butyl-DL-2-(2-naphthyl)-2-hydroxyacetic acid methyl ester

100 mmole of DL-2-(2-naphthyl)-2-hydroxyacetic acid methyl ester(Example 13, Step A) is dissolved in 100 ml of methylene chloride. Thesolution is cooled to -25° C., 0.88 ml (10 mmole) oftrifluoromethanesulfonic acid and 116-120 ml of isobutylene are addedand the mixture is left to stand at -20° C. for 30 minutes. Then 1.0 mlof pyridine is added and the reaction mixture is left to warm up to roomtemperature, then it is evaporated from a water bath at about 40° C. and20-25 millibar. The evaporation residue is dissolved in 100 ml ofbenzene, washed with 20 ml portions of water, 5% sodium hydrogencarbonate solution, water, 1M potassium hydrogen sulfate and again withwater, then dried over sodium sulfate and evaporated from a water bathat about 40° C. and 20-25 millibar. The residue is considered 100 mmoleof O-t-butyl-DL-2-(2-naphthyl)-2-hydroxyacetic acid methyl ester andused in Step C R_(f) (12)=0.50-0.55!.

Step C: O-t-Butyl-DL-2-(2-naphthyl)-2-hydroxyacetic acid

100 mmole of O-t-butyl-DL-2-(2-naphthyl)-2-hydroxyacetic acid methylester (Example 13, Step B) is dissolved in 100 ml of methanol, 50 ml of2M sodium hydroxide solution is added and the solution is stirred atroom temperature overnight. The reaction mixture is saturated withcarbon dioxide by adding dry-ice, washed with 2×20 ml of n-hexane,acidified with solid potassium hydrogen sulfate and extracted with 3×40ml of ethyl acetate. The ethyl acetate layers are combined, washed toneutrality with water, dried over sodium sulfate and evaporated from awater bath at about 40° C. and 20-25 millibar. The oily residue is mixedwith 100 ml of n-hexane, the crystals formed are filtered, washed withn-hexane and dried at reduced pressure.

Yield 13.2 g (51%); m.p.: 114°-116° C.

R_(f) (9)=0.34-0.38

Analysis for C₁₆ H₁₈ O₃ (258.30) Calculated: C %=74.39; H %=7.02; Found:C %=74.0 ; H %=7.0

Step D: O-t-Butyl-DL-2-(2-naphthyl)-2-hydroxyacetic acid2,4,5-trichlorophenyl ester

12.9 g (50 mmole) of O-t-butyl-DL-2-(2-naphthyl)-2-hydroxyacetic acid(Example 13, Step C) and 10.24 g (52 mmole) of 2,4,5-trichlorophenol aredissolved in 30 ml of dimethyl formamide and cooled to 0° C. 10.3 g (50mmole) of dicyclohexylcarbodiimide is added and the mixture is stirredat room temperature for 2 hours. The precipitated dicyclohexylurea isfiltered off and washed with 2×5 ml of dimethyl formamide, then thefiltrate and the washings are combined. The solution obtained isconsidered 50 mmole of O-t-butyl-DL-2-(2-naphthyl)-2-hydroxyacetic acid2,4,5-trichlorophenyl ester R_(f) (9)=0.82-0.85! used in the nextreaction step as a solution in dimethyl formamide.

Step E: O-t-Butyl-DL-2-(2-naphthyl)-2-hydroxyacetyl-L-proline

To the dimethyl formamide solution ofO-t-butyl-DL-2-(2-naphthyl)-2-hydroxyacetic acid 2,4,5-trichlorophenylester (50 mmole), obtained in Example 13, Step D, 5.8 g (50 mmole) ofL-proline and 7.0 ml (50 mmole) of triethylamine are added, then themixture is stirred at room temperature for 24 hours. The reactionmixture is filtered and evaporated from a water bath at about 40° C. and20-25 millibar. Thereafter 60 ml of 5% sodium hydrogen carbonate isadded to the oily residue. The cloudy solution is washed with 3×40 ml ofdiethyl ether, the pH is adjusted to 3 by adding solid potassiumhydrogen sulfate, then the solution is extracted with 3×40 ml of ethylacetate. The combined ethyl acetate solutions are washed to neutralitywith water, dried over sodium sulfate and evaporated from a water bathat about 40° C. and 20-25 millibar. The resulting 18 g oily residue isconsidered the diastereomer mixture ofO-t-butyl-D-2-(2-naphthyl)-2-hydroxyacetyl-L-proline 25 mmole; R_(f)(1)=0.38-0.40! and O-t-butyl-L-2-(2-naphthyl)-2-hydroxyacetyl-L-proline25 mmole; R_(f) (1)=0.35-0.37! and is used in this oil form in the nextstep (F).

Step F: O-t-Butyl-D-2-(2-naphthyl)-2-hydroxyacetyl-L-proline

2000 ml of BIO-RAD AG 1-X2 (50-100 mesh) anion-exchange resin (acetatecycle, anion capacity 1200 mequiv.) is suspended in distilled water,poured into a glass column (80×6 cm), then 4000 ml of a mixture ofmethanol and water (2:1) is run through it at a flow rate of 15 ml/min.Thereafter the solution of 18 g of an oil, prepared in Example 13, StepE, dissolved in 2000 ml of a mixture of methanol and water (2:1), isapplied on it, then the resin is washed with 4000 ml of the abovemixture of methanol and water. A mixture of methanol and 1M acetic acid(2:1) is used for eluting the product, collecting 200 ml fractions. Thefractions are monitored by thin-layer chromatography in the system ofethyl acetate:pyridine:acetic acid:water (480:20:6:11), the spots arevisualized under UV light or by developing in KMnO₄. The fractionscontaining the pure isomers are combined and concentrated to a thirdvolume on a water bath at about 40° C. and 20-25 millibar. Theprecipitated product is redissolved by the addition of acetonitrile andthe solutions are frozen and freeze-dried. Yields:

Isomer I R_(f) (1)=0.38-0.40! 5.93 g

Isomer II R_(f) (1)=0.35-0.37! 5.63 g.

Mixture of isomers I and II 1.0 g.

On the basis of the chromatographic properties and enzyme inhibitoryactivities of the aldehyde derivatives prepared from them, isomer I isO-t-butyl-D-2-(2-napthyl)-2-hydroxyacetyl-L-proline 5.93 g (16.68mmole), 66.7% (calculated for the intermediary productO-t-butyl-DL-2-(2-naphthyl)-2-hydroxyacetic acid, used in Example 13,Step D)!, m.p.: 90°-93° C.

Analysis for C₂₁ H₂₅ NO₄ (355.42) Calculated: C %=70.96; H %=7.09; N%=3.94; Found: C %=71.0; H %=7.15; N %=3.85.

EXAMPLE 14 D-2-(1-Naphthyl)-2-hydroxyacetyl-L-prolyl-L-arginine aldehydehemisulfate

Step 1: O-t-Butyl-D-2-(1-naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

Starting from 3.12 g (8 mmole) of t-butyloxycarbonyl-N^(G)-benzyloxycarbonyl-L-arginine lactam S. Bajusz et al.: J. Med. Chem. 33,1729 (1990)! and 2.8 g (7.8 mmole) ofO-t-butyl-D-2-(1-naphthyl)-2-hydroxyacetyl-L-proline (Example 14, StepF) and proceeding according to the process described in Example 13, Step1, applying identical amounts of solvents and reagents, 4.14 g (85%) ofthe product is obtained.

R_(f) (9)=0.71-0.77

Step 2: D-2-(1-Naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam

Transforming 4.08 g (6.5 mmole) ofO-t-butyl-D-2-(1-naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 14, Step 1) according tothe process described in Example 13, Step 2, applying identical amountsof solvents and reagents, 1.85 g (50%) of the product is obtained.

R_(f) (9)=0.44-0.46.

Analysis for C₃₁ H₃₃ N₅ O₆ (571.61) Calculated: C %=65.13; H %=5.82; N%=12.25; Found: C %=65.0 H %=5.95 N %=12.0.

Step 3: D-2-(1-Naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde

Transforming 1.71 g (3 mmole) ofD-2-(1-naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine lactam (Example 14, Step 2) according tothe process described in Example 13, Step 3, applying identical amountsof solvents and reagents, 1.2 g (70%) of the product is obtained.

R_(f) (3)=0.40-0.44

Step 4: D-2-(1-Naphthyl)-2-hydroxyacetyl-L-prolyl-L-arginine aldehydehemisulfate

Transforming 1.0 g (1.75 mmole) ofD-2-(1-naphthyl)-2-hydroxyacetyl-L-prolyl-N^(G)-benzyloxycarbonyl-L-arginine aldehyde (Example 14, Step 3) according tothe process described in Example 13, Step 4, applying identical amountsof solvents and reagents, 0.8 g (90%) of the product is obtained.

R_(f) (5)=0.28-0.30

HPLC(I/3): k'=5.99, 6.39 and 7.17.

The FAB mass spectrum confirms the assumed structure (440 M+H!⁺).

The starting materials can be prepared as follows:

O-t-Butyl-D-2-(-naphthyl)-2-hydroxyacetyl-L-proline

Step A: DL-2-(1-naphthyl)-2-hydroxyacetic acid methyl ester

Starting from 20.2.g (100 mmole) of DL-2-(1-naphthyl)-2-hydroxyaceticacid I. M. Panaiotov: Compt rend. acad. bulgare sci. 10, No. 2, 137-140(1957); Chem. Abstr. 52, 5336e (1958); m.p.: 92°-94° C.; R_(f)(2)=0.28-0.30! and proceeding according to the process described inExample 7, Step B, applying proportional amounts of solvents andreagents, an oily product R_(f) (2)=0.69-0.71!. is obtained which isconsidered 100 mmole of DL-2-(1-naphthyl)-2-hydroxyacetic acid methylester.

Step B: O-t-Butyl-DL-2-(1-naphthyl)-2-hydroxyacetic acid methyl ester

Starting from 100 mmole of DL-2-(1-naphthyl)-2-hydroxyacetic acid methylester (Example 14, Step A) and proceeding according to the processdescribed in Example 13, Step B, applying identical amounts of solventsand reagents, 100 mmole of O-t-butyl-DL-2-(1-naphthyl)-2-hydroxyaceticacid methyl ester is obtained and applied in the next step.

R_(f) (12)=0.66-0.70.

Step C: O-t-Butyl-DL-2-(1-naphthyl)-2-hydroxyacetic acid

Starting from 100 mmole of O-t-butyl-DL-2-(1-naphthyl)-2-hydroxyaceticacid methyl ester (Example 14, Step B) and proceeding according to theprocess described in Example 13, Step C, applying identical amounts, ofsolvents and reagents, 14.6 g (56.5%) of the product is obtained.

R_(f) (9)=0.73-0.76; m.p.: 132°-135° C.

Analysis for C₁₆ H₁₈ O₃ (258.30) Calculated: C %=74.39; H %=7.02; Found:C %=74.3; H %=7.05.

Step D: O-t-Butyl-DL-2-(1-naphthyl)-2-hydroxyacetic acid2,4,5-trichlorophenyl ester

Starting from 12.9 g (50 mmole) ofO-t-butyl-DL-2-(1-naphthyl)-2-hydroxyacetic acid (Example 14, Step C)and 10.24 g (52 mmole) of 2,4,5-trichlorophenol and proceeding accordingto the process described in Example 13, Step D, applying identicalamounts of solvents and reagents, a dimethyl formamide solution isobtained which is considered 50 mmole ofO-t-butyl-DL-2-(1-naphthyl)-2-hydroxyacetic acid 2,4,5-trichlorophenylester R_(f) (9)=0.84-0.86! and is directly applied in the next step.

Step E: O-t-Butyl-DL-2-(1-naphthyl)-2-hydroxyacetyl-L-proline

Starting from 50 mmole of O-t-butyl-DL-2-(1-naphthyl)-2-hydroxyaceticacid 2,4,5-trichlorophenyl ester (Example 14, Step D) and 5.8 g (50mmole) of L-proline and proceeding according to the process described inExample 13, Step E, applying identical amounts of solvents and reagents,15.65 g of an oily residue is obtained R_(f) (1)=0.53-0.55); HPLC(II/5):k'=12.85 and 16.95! which is considered the mixture of 22 mmole ofO-t-butyl-D-2-(1-naphthyl)-2-hydroxyacetyl-L-proline and 22 mmole ofO-t-butyl-L-2-(1-naphthyl)-2-hydroxyacetyl-L-proline and is used in thisoil form in the next (F) step.

Step F: O-t-Butyl-D-2-(1-naphthyl)-2-hydroxyacetyl-L-proline

The 15.65 g of oily product, obtained in Example 14, Step E, issubmitted to chromatography according to the method applied in Example13, Step F on the anion-exchange resin BIO-RAD AG 1-X2 (acetate cycle),except that the content of the fractions is assayed by HPLC underconditions specified in Step E (II/5). The following products areobtained:

Isomer I 4.4 g (k'=12.84) eluted first

Isomer II 4.3 g (k'=16.95) eluted last.

Mixture of isomers I and II 4.5 g.

On the basis of the chromatographic properties and enzyme inhibitoryactivities of the aldehyde derivatives prepared from them, isomer I isO-t-butyl-D-2-(1-napthyl)-2-hydroxyacetyl-L-proline 4.4 g (12.4 mmole),49.6 % (calculated for the intermediary productO-t-butyl-DL-2-(1-naphthyl-2-hydroxyacetic acid, used in Example 14,Step D); m.p.: 88°-90° C.!.

Analysis for C₂₁ H₂₅ NO₄ (355.42) Calculated: C %=70.96; H %=7.09; N%=3.94; Found: C %=70.95; H %=7.15; N %=3.8.

What we claim is:
 1. A peptide derivative selected from the groupconsisting ofD-isochroman-1-carbonyl-L-prolyl-L-arginine aldehyde,L-isochroman-1-carbonyl-L-prolyl-L-arginine aldehyde,D-isochroman-3-carbonyl-L-prolyl-L-arginine aldehyde,L-isochroman-3-carbonyl-L-prolyl-L-arginine aldehyde,D-2-phenyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehyde,2-(9-fluorenyl)-2-hydroxyacetyl-L-prolyl-L-arginine aldehyde,3-cyclohexyl-D-lactyl-L-prolyl-L-arginine aldehyde,D-2-phenyl-2-hydroxyacetyl-L-pipecolyl-L-arginine aldehyde,D-2-cyclohexyl-2-hydroxyacetyl-L-pipecolyl-L-arginine aldehyde,3-cyclohexyl-D-lactyl-L-pipecolyl-L-arginine aldehyde, and3,3-diphenyl-D-lactyl-L-prolyl-L-arginine aldehyde and the acid-additionsalts of these compounds.
 2. 3-Cyclohexyl-D-lactyl-L-prolyl-L-argininealdehyde hemisulfate.
 3. 3,3-Diphenyl-D-lactyl-L-prolyl-L-argininealdehyde hemisulfate.